Screening assays and methods

ABSTRACT

Screening assays and methods of performing such assays are provided. In certain examples, the assays and methods may be designed to determine whether or not two or more species can associate with each other. In some examples, the assays and methods may be used to determine if a known antigen binds to an unknown monoclonal antibody.

RELATED U.S. APPLICATION

[1] This application is a continuation of U.S. patent application Ser.No. 12/857,508 filed Aug. 16, 2010, which is a divisional of U.S. patentapplication Ser. No. 11/523,124 filed Sep. 18, 2006, which claims thebenefit of U.S. provisional application No. 60/717,976 filed Sep. 16,2005, each of which is incorporated by reference herein in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was funded in part by the U.S. Government under grantnumbers NAKFI Nano08 awarded by the National Academy of Sciences and/orgrant 5R01AI034893-1 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE TECHNOLOGY

The invention relates to screening assays and methods to identifysecreted products.

BACKGROUND

Assays exist to identify compounds or molecules of interest that may beinvolved in a disease process or other condition or in treating adisease process or condition. Existing assays have some drawbacks. Onesignificant drawback is the time required to screen for many compoundsor molecules. Another drawback is that it may not be possible to recoverthe compound or molecule post-screening. There remains a need for betterscreening assays and methods.

SUMMARY

The invention provides a printed microarray of unknown cell-derivedproducts. Each position of the printed array comprises a deposit, whichis less than 100 micrometers in diameter, and corresponds to a secretionof a single cell. The deposit is a secreted product such as an antibody,cytokine, chemokine, or inflammatory mediator or another cellularcomponent, e.g., DNA, RNA, or a lipid, which is liberated from an intactcell upon lysis or permeabilization of the cell. Optionally, the printedmicroarray comprises a capture ligand, e.g., which binds to a singleimmunoglobin isotype or a chemical capture moiety, e.g., a supportderivatized to retain a class of secreted products.

An engraving plate includes a plurality of wells, each of the wells isless than 100 micrometers in diameter and comprises a single cell.Preferably, the number of cells is less than 5 cells. The engravingplate is a gas-permeable conformable composition. The plate has anelastic modulus (Young's Modulus) in the range of 200-2000 Kilopascal(kPa). The composition of the plate is preferablypoly(dimethylsiloxane). The wells of the plate contain at least onecell. That cell is an immune cell, an antibody-producing cell, ahybridoma cell, a T cell, or other cell from the blood or a tissue. Thefunction or secretory profile of the cell or cells is unknown. The cellproduces a recombinant secreted polypeptide, the polypeptide has anamino acid target sequence for a chemical modification or a recombinantimmunoglobulin chain that has an amino acid sequence of an enzymecleavage site.

This invention also provides a method of screening by disposing aconformable support comprising a plurality of uncharacterized secretorycells on a substrate, e.g., a glass slide, a plastic slide or a bead andexposing a first species comprising an unknown cell-derived, e.g.,secreted, product, transferred from the conformable support to thesubstrate, to a second species to determine if the first species and thesecond species associate. The second species is a known target ligand,e.g., a defined antigen of a pathogenic organism. For example, the firstspecies is an antibody and the second species is a defined targetantigen for which antibody binding is sought.

Another method comprises depositing a suspension of cells onto amoldable slab containing at least one microwell that forms a microwellarray that allows the suspension of cells to settle where at least onecell settles into the at least one microwell of the microwell array. Themicrowell array then contacts a substrate, which is pretreated with afirst species. The microwell array is then incubated, for about 1, 5,10, 20, 30, 40, 50 min but less than 24 hours, and allowing at least onecell to secrete a second species. After incubating, the first speciesand the second species form an association of the substrate, which iswhere the microarray is formed. The microarray is then removed from themoldable slab, which still contains cells in the microwells and isplaced in a reservoir containing a medium. The cells in the microwellscan be maintained in the medium and the microwell array can contact anew substrate and form more than a new microarray, whereby the microwellarray can “stamp” more than one microarrays, where about 5 to about 100microarrays are formed. The association is then detected between thefirst species and the second species on the microarray.

The second species, which is secreted by the cells, is a monoclonalantibody or a cytokine and the first species is a secondary antibody,wherein a labeled antigen, or fragment thereof, can associate with themonoclonal antibody. Or the first species is an antigen and the secondspecies is a monoclonal antibody, wherein a labeled secondary antibodycan associate with the monoclonal antibody. The label is a fluorescentlabel, a colorimetric label or a radio label. The association on themicroarray can be detected with at least one labeled species. The cellis a bacterial cell or a hybridoma, which then is retrieved from themoldable slab if an association occurs between the first species and thesecond species. The cell is challenged with an antigen prior to disposalof the moldable slab on the substrate. The second species also comprisesa catalyst, which is an enzyme and the first species is a potentialenzyme substrate or a potential enzyme substrate analog.

The moldable slab is fabricated by soft lithography and replica moldingand is of a biocompatible material, which is not toxic and gaspermeable. The moldable slab, made of poly(dimethylsiloxane), cancompress against the substrate to form a tight, but reversible seal withthe substrate. The microwell array comprises a block of wells where awell has a diameter of about 50 μm and a depth of about 50 μm and thewells are separated by about 50 μm or a well has a diameter of about 100μm and a depth of about 100 μm and the wells are separated by about 100μm. The wells are sized to retain about 1 nanoliter or less of fluid.

The invention provides a method of screening a monoclonal antibody bycontacting a moldable slab with a substrate, which has at least onesecondary antibody. The moldable slab contains at least one microwelland at least one hybridoma that secretes a monoclonal antibody in the atleast one microwell. The monoclonal antibody is then exposed to at leastone antigen to determine if the monoclonal antibody can bind to theantigen. The method is performed in less than about one day or about 6hours.

A method of screening a monoclonal antibody by contacting a moldableslab to a substrate, where the moldable slab has at least one microwelland at least one hybridoma that secretes a monoclonal antibody in themicrowell. The substrate has at least one antigen or at least onesecondary antibody on a surface of the substrate. The microarray formedis then used to detect if the monoclonal antibody can bind to the atleast one antigen or the at least one antibody on the surface of thesubstrate. The method of screening the monoclonal antibody is performedin less than about one day or about 6 hours.

A kit is assembled that comprises a substrate, a moldable slabconfigured to receive the substrate and to provide a fluid tight sealbetween the moldable slab and the substrate, and instructions for usingthe conformable support and the substrate to identify species that mayassociate. The moldable slab or the substrate or both of the kitcomprises one or more materials selected from the group consisting ofglass, plastic, polystyrene, polycarbonate, poly(dimethylsiloxane),nitrocellulose, poly(vinylidene fluoride), or a metal. The metal is oneor more of gold, palladium, platinum, silver, steel or alloys ormixtures thereof. The substrate is a glass slide, a plastic slide or abead and the moldable slabs contains a microwell array.

A kit comprising a substrate, a moldable slab having a plurality ofmicrowells and configured to receive the substrate and to provide afluid tight seal between the moldable slab and the substrate, andinstructions for using the moldable slab and the substrate to identifyspecies that may associate.

A test apparatus comprising a moldable slab comprising at least onemicrowell that forms a microwell array that contacts a substrate in amanner to provide a fluid tight seal between the moldable slab and thesubstrate. The apparatus puts one species, generally a cell, in at leastone well of the microwell array and the microwells of the moldable slabare sized and arranged to retain about one nanoliter or less of fluidvolume. The analytical methods described herein offer numerousadvantages over prior methods, including time-saving and costeffectiveness.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a top view and FIG. 1B is a cross-section of a schematicalong section line 1B-1B of a moldable slab, in accordance with certainexamples.

FIG. 1C is a top view of an insert and FIG. 1D is a side view of aninsert in contact with a moldable slab, in accordance with certainexamples.

FIGS. 2A-2D are schematics of a method for transferring materialsecreted by a cell to a substrate, in accordance with certain examples.

FIGS. 3A-3D are schematics of a method for detecting association of twospecies, in accordance with certain examples.

FIGS. 4A-4D are schematics of a membrane and its use with one or moresubstrates, in accordance with certain examples.

FIG. 5 is a photograph of Hyb9901 cells (anti-ovalbumin) disposed in 100micron diameter microwells molded in poly(dimethylsiloxane), inaccordance with certain examples.

FIGS. 6A-6E are schematics of a method for immobilizing antibodies on asubstrate, in accordance with certain examples.

FIG. 7 is a graph showing the percentages of wells filled by one or morecells versus the concentration of cells, in accordance with certainexamples.

FIG. 8 is a graph showing the average number of cells per well versusthe concentration of cells, in accordance with certain examples.

FIG. 9 is a bar graph showing the number of cells counted per well fortwo different concentrations of cells, in accordance with certainexamples.

FIG. 10 is a graph showing the percent of viable cells as a function ofincubation time, in accordance with certain examples.

FIG. 11A, is a micrograph of Hyb9901 cells disposed in 100 microndiameter wells composed of polydimethylsiloxane and FIG. 11B is amicrograph of the slide, on which the array of microwells of FIG. 11Awere disposed for 4 h, with physisorbed ovalbumin that associated withmouse anti-ovalbumin and was probed with a fluorescent secondaryantibody (Goat-anti Mouse), in accordance with certain examples.

FIG. 12A is a micrograph of Hyb9901 cells disposed in 100 microndiameter wells composed of polydimethylsiloxane and FIG. 12B is amicrograph of physisorbed Protein G and secondary Ab (Goat-anti Mouse)on a glass slide, incubated with microwells of FIG. 12A for 4 hours(anti-ovalbumin hybridomas), after the glass slide was probed withfluorescent antigen (Ovalbumin-Alexa 488), in accordance with certainexamples.

FIGS. 13A-D is a schematic diagram depicting method for preparation ofengraved arrays of secreted products from a mixture of cells.

FIGS. 14A-E is a schematic displaying two methods for detection ofantibodies on the surface of a substrate after microengraving.

FIGS. 15A-B are fluorescence micrographs of two microarrays preparedsequentially using the same array of microwells.

FIG. 16A is a fluorescence micrograph of a region of a microarraygenerated from a polyclonal mixture of cells and a phase contrastmicrograph.

FIG. 16B is an autoradiograph of ³⁵S-labelled H-2K^(b)immunoprecipitated using supernatants from cultures containing Hyb099-01, Y3, and four clones.

FIG. 16C is a fluorescence micrograph of a region of a microarrayshowing conjugation of captured antibody.

FIG. 17A is a micrograph showing spots generated from a polyclonalmixture of hybridomas that are reactive with fluorescently labeledH-2K^(b) tetramers.

FIG. 17B is a micrograph showing spots generated from one expandedhybridoma (clone 136) that are reactive with fluorescently labeledH-2K^(b) tetramers (red) and goat-anti-mouse IgG (green).

FIG. 18 is a drawing of a schematic illustration of proposed method foridentifying antibodies that bind surface-expressed epitopes on apathogen.

FIG. 19A is a graphic of the capture of different serotypes of the samemicrobe.

FIG. 19B is a drawing of the capture of altered microbe (genetic mutant,drug, enzyme-treated) to discover rare epitopes.

FIG. 20 is a fluoresence micrograph of human IgG captured bymicroengraving with EBV-transformed B cells and labeled with Alexa488-goat-anti-human IgG.

FIG. 21 is a drawing of a scheme for generating a single assay tomeasure both the phenotype and secretory function of individual cells.

FIG. 22 is a drawing of a schematic diagram depicting the process fordepositing cells from a suspension into microwells and capturingantibodies on a solid support.

FIG. 23 is a fluorescent micrograph of an engraved microarray stainedfor captured IFNγ (green) and IL-4 (red, indicated with arrowheads).

FIG. 24 is a drawing of a graphical abstract of antibodies engineered toallow enzymatic installation of a specific chemical moiety that canreact with a functionalized organic surface designed to resistnon-specific adsorption of proteins.

FIG. 25 is a drawing of a method for attaching antibodies modified withan analog of biotin to a SAM bearing a reactive hydrazide moiety.

It will be recognized by the person of ordinary skill in the art, giventhe benefit of this disclosure, that the examples shown in the figuresare not necessarily drawn to scale. Certain features or components mayhave been enlarged, reduced or distorted to facilitate a betterunderstanding of the illustrative aspects and examples disclosed herein.In addition, the use of shading, patterns, dashes and the like in thefigures is not intended to imply or mean any particular material ororientation unless otherwise clear from the context.

DETAILED DESCRIPTION

The methods of the invention allow identification and characterizationof previously unknown or undefined cell-derived compositions. Thecompositions are secreted such as antibodies or cytokines orcompositions that are released upon lysis or permeabilization of thecell such as cytoplasmic, nuclear, or cell membrane components. Anycellular composition including biopolmers, e.g., DNA and RNA, as well aslipids and glycolipida, is captured by this method provided that thesolid support (substrate) is appropriately functionalized (e.g., poly dTnucleotides for RNA). In addition to immune cells, other eukaryoticcells are interrogated. For example, single cell suspensions from normalor cancerous tissue of tissue type, e.g., heart, brain, liver, prostate,breast, or colon. The systems are also useful to characterize wholecells and secreted products from their cell types (bacteria, yeast,small parasites (malaria)).

In addition to analysis of secreted cell-derived products, the methodencompasses characterization of the cells remaining in the wells afterthe capture of the secreted products (e.g., by immunofluoresence,genetic sequencing). In conjunction with identification andcharacterization of secreted products, the cells in corresponding wellsare retrieved and matched with the materials secreted.

The methods, apparatus and kits disclosed herein are used to identifyantibodies and other cell secreted products. Hybridomas may beidentified using these methods. Hybridomas are made using known methods,for example, a mouse is immunized with an antigen or a plurality ofantigens. The spleen of the mouse is removed and broken up to form asuspension. The suspended spleen cells are fused with mouse myelomacells, e.g., in polyethylene glycol. The cells are then cultured overseveral days in media containing hypoxanthine, aminopterin and thymidine(HAT media) such that any unfused spleen cells die. Unfused myelomacells also are killed in the HAT media because they lack the enzymehypoxanthine ribosyl transferase (HPRT) and thus cannot produce inosinefrom hypoxanthine, and the aminopterin prevents the myeloma cells fromusing an alternative pathway to produce inosine from thymidine. Becauseinosine is a precursor to many nucleic acid pyrimidines, the myelomacells die because they are unable to produce nucleic acids. In contrast,the hybridomas have the HPRT and can use the hypoxanthine and thethymidine in the HAT media to produce nucleic acids.

Using the methods, apparatus and kits disclosed herein, a single, or afew hybridomas are placed in a microwell of a moldable slab and areallowed to produce monoclonal antibodies for a few hours, e.g., 4-8hours. Secreted monoclonal antibodies are immobilized on a substrate andexposed to known antigen(s) to determine which monoclonal antibodiesbinds to the known antigen. Results lead to a rapid high-throughputscreening of hybridoma cells that product antibodies against specificantigens and also allows for the identification and expansion of cellsproducing monoclonal antibodies.

Another application for the method, apparatus and kits disclosed hereinis to identify personalized antibodies raised against tissue samplesfrom an individual. For example, the methods, apparatus and kitsdisclosed herein may be used to raise antibodies against a tumor in anindividual. A sample or biopsy of the individual's tumor may be takenand injected into a mouse. After several weeks, the mouse's spleen cellsmay be removed and monoclonal antibodies may be identified. Productionof such monoclonal antibodies may provide for treatment of theindividual's tumor by injection or administration of the producedmonoclonal antibodies, e.g., site-specific delivery of the monoclonalantibodies. Fully humanized antibodies containing a human constantregion (Fc) would be ideal for this application.

Another application for the method, apparatus and kits disclosed hereinis to determine the efficacy of an immune response related to aparticular immunotherapy such as a vaccine. For example, the number oflymphocytes secreting specific molecules indicative of proliferation(e.g., TNF-alpha, IL-1, IgE) and the overall output levels of thesemarkers from individual cells may be assayed. The assay may requireantibodies against each secreted factor immobilized on the substrate tocapture molecules produced by individual cells and a second specificantibody with a fluorescent marker to screen for the amount of eachmarker captured on the substrate. This assay produces a new type ofpersonalized medicine for evaluating the responsiveness of individualsto selected immunotherapies.

The advent of antimicrobial drugs and vaccines using inactivated orattenuated microorganisms has had a remarkable impact on the overallhealth of the world population in the last 150 years, especially infirst-world countries. In the present era, however, there has been aresurgence of infectious diseases thought to be under control oreradicated. New outbreaks of lethal infectious diseases that have beensuppressed in recent times by the administration of antibiotics or firstgeneration vaccines (staphylococcus, rubella, mumps) underscore thisobservation. Evolutionary pressure applied to various microorganisms bythe use of vaccines and antimicrobial drugs has resulted in increasingnumbers of strains resistant to current therapeutics. These factorscombined with both existing and potential epidemic diseases—HIV,malaria, influenza—for which treatments are limited, if available atall, suggest a need for new therapies—either vaccines or drugs.

Two current strategies for developing new therapies for infectiousdiseases are: 1) rational design of vaccines, and 2) generation oftherapeutic monoclonal antibodies (mAb). The knowledge of the genomesfor common pathogens, and the advent of computational tools for miningthese data, has enabled a more rational approach for determiningimmunogenic factors than empirical data from inactivated materials.Although polyclonal sera containing antibodies have been used clinicallyfor passive immunization since the late 1800s, their use in modernmedicine has been limited by the advent of antimicrobial drugs. Thedevelopment of hybridomas and related technologies in the 1970s and1980s has led to monoclonal therapies, but economic and politicalsuasions have focused these treatments largely on cancer and autoimmunedisorders. The threat of bioterrorism has renewed interest inidentifying antibodies against potential biological agents, e.g.,anthrax.

At least two factors continue to hinder progress in identifyingsurface-expressed, immunogenic epitopes on pathogens and subsequently,new therapies. First, genomic analysis has yielded some leads forformulating new vaccines, but it can not predict other factors that canenhance (or mask) the immunogenicity of an epitope—post-translationalmodifications, conformational variations, non-proteinaceous materials,or genetic variation among serotypes. Second, for therapies based onneutralizing antibodies, it is likely that single mAbs for infectiousdiseases will be insufficient; a cocktail of mAbs recognizing a range ofepitopes should be more effective than a single mAb. Libraries ofsuitable mAbs from which to create such cocktails are small ornon-existent. Thus, new tools for the rapid identification of antigensthat evoke robust and protective immune responses for a large number ofinfectious agents would greatly assist in the design of vaccines againstepidemic infectious diseases (malaria, HIV, influenza) and prophylactictreatments for others (small pox, anthrax). This screening technologyenables high-throughput and rapid analysis of large polyclonalpopulations of immortalized B cells (>100,000 cells in <12 h) toidentify clones producing antibodies reactive against the surfaceepitopes of a pathogen.

There is growing evidence that common phenotypic markers (e.g., CD4+)can encompass a number of subsets of cells with diverse functions,indicated by the types of cytokines secreted. A simple analytical toolthat provides information about both phenotypic markers and secretedfactors for individual primary cells (without extended ex vivoculturing) facilitates studies in cell biology, especiallyimmunobiology. The methods disclosed make it possible to retrieve thecells for subsequent culture or genetic analysis, applications includei) profiling immunological responses to administered vaccines, allergicreactions, or foreign pathogens, and ii) extending understanding of thecell biology of cancers and autoimmune disorders.

Also, the disclosed method, apparatus and kits is a simple analyticalassay for individual primary cells that allows both determination oftheir phenotypes (expressed surface or intracellular proteins) anddirect measurement of their functional behaviors (secreted cytokines,antibodies, growth factors). More specifically, the technology rapidlycorrelates surface-displayed phenotypes with functional secretorybehaviors of individual primary cells, preferably without extendedculture or other manipulations that could modify the expression ofmarkers or the behavior. Application of the methods, apparatus and kitsmake it possible to have a platform for the systematic analysis of animmune response to various diseases, allergies, and treatments (e.g.,vaccines) or a systematic analysis of immune responses for individualsto various diseases using limited sizes of samples, and shouldfacilitate the transition of clinical medicine towards predictive,personalized healthcare.

Antibodies are ubiquitous reagents in biology for applications thatrange from basic biochemistry to clinical diagnostics. They are commonlyimmobilized on surfaces to retrieve other biopolymers from a surroundingsolution (sera, culture supernatant). Examples of supports used includebeads/resins, microarrays, and nanoparticles. The most frequent methodsfor immobilizing antibodies are i) physical adsorption onto ahydrophobic substrate, ii) covalent attachment at reactive sites on theprotein, or iii) non-covalent interactions between an immobilizedreceptor (streptavidin, protein A or G, anti-Ig) and an antibody or itsderivative multiply decorated with ligands. These strategies do notinduce a specific orientation on every antibody immobilized—for example,with the binding region positioned away from the underlying surface.Though random orientation of a protein on surfaces may be sufficient inmany assays designed to determine unknown protein-protein interactions,the development of miniaturized biological assays that incorporatemicro- and nanoscale components (with limited surface areas) motivatesthe need for new strategies to attach antibodies (and other proteins) onsurfaces that preserve function in high-density. The sensitivity ofdetection and the yield of capture depend on the number of binding sitesavailable at the interface between the supporting surface and thesurrounding solution. Two key parameters influencing this value are: i)the density of molecules and ii) the accessibility of the binding regionon the immobilized molecule to other molecules at the interface.Orientation of an immobilized molecule on the surface is, therefore,important for improving accessibility.

The miniaturization of microarrays on planar surfaces can improve thedensity of information available in a single experiment, and theincorporation of nanoparticles in biological assays can improve thesensitivity of diagnostics. Both applications require functional organicsurfaces capable of binding specific molecules from a surroundingsolution, but in both instances, the available surface area per featureor particle is limited. This characteristic suggests that the overalllimits of detection afforded by these methods will depend on the numberof functional receptors presented at the interface between the solidsupport and the surrounding environment. Although antibodies are usedroutinely in immunochemical assays for detecting specific analytes, mosttechniques for immobilizing them on surfaces do not favor a particularorientation of the molecules. This example outlines an approach forengineering full-length antibodies to carry a specific chemicalfunctionality at the C-terminus of the heavy chains of theimmunoglobulin. The addition of a short peptide sequence recognized byan enzyme, BirA ligase from Escherichia coli, makes it possible toincorporate an unnatural analog of biotin containing a ketone to theantibody. Reaction of this moiety with an organic surface designed toresist non-specific adhesion of other proteins improves the orientationof the modified antibodies attached to the surface. This generalapproach extends to other proteins of interest (fragments of antibodies,recombinant enzymes).

Examples of the technology disclosed herein may be used to identifyunknown species disposed on a substrate that can associate with a knownspecies. For examples, the methods disclosed herein are used to identifyan antibody that binds to or interacts with a desired target antigen.The exact nature of the assays depends, for example, on the selectedspecies, the selected slab or substrate and the information desired fromthe assay. Certain embodiments of the technology disclosed hereinprovides significant advantages including, for example, (1) a singlearray of microwells can contain greater than 625 wells per square inchcompared to about 1 well per square inch for a conventional 96-wellplate; (2) the dilution of a single cell per well makes it possible toidentify cells producing antigen-specific antibodies in a single screencompared to iterative testing required for assays using conventionalmethods, such as a 96- or 384-well plates; (3) the limited volume of themicrowells (about 1 nanoliter or less) permits sufficient concentrations(e.g., about 1 μM) of antibody to be reached within a few hours insteadof 5-7 days; (4) an assay for positive antibodies may be integrated intoa method and does not require any additional manipulations to array ortest the secreted antibodies for specificity; and (5) the screeningmethods, apparatus and kits can be multiplexed to screen simultaneouslyfor many different cells producing antibodies against differentantigens.

In accordance with certain examples, the methods, apparatus and kitsdisclosed herein may be used in determining whether or not two speciesassociate. As used herein, the term “associate” refers to interactionssuch as binding, adsorption, ionic attraction or some other type ofinteraction between the two species. In some examples, species thatassociate preferably bind to each other with an association constant ofat least about 10⁹ M⁻¹ or larger. Species which bind to each other withsuch association constants allow for easy distinction between speciesthat associate and those that do not associate.

In accordance with certain examples, a moldable slab may be used in themethods and kits described herein. As used herein “moldable slab” refersto an apparatus which can flex, move or distort, at least in onedimension, when placed in contact with a substrate. For example, incertain configurations the moldable slab may include a material, e.g.,an elastomeric material, such that as the moldable slab is placed incontact with a substrate, a substantially fluid tight seal may be formedbetween the moldable slab and the substrate to retard or to prevent anyfluid in the moldable slab from escaping or leaking.

Protocols for identifying cells from a single colony that serve assources of monoclonal antibodies rely on limited dilution of candidatecells into microtiter plates. The cells are diluted into 96-well platesor 384-well plates, and expanded for 5-7 days. At that time, aliquots ofthe media from each well may be tested to identify positive wellsproducing the desired antibody. For example, an immunoassay such asenzyme-linked immunosorbant assay (ELISA) may be used to identifypositive wells producing the desired antibody. The contents of thepositive well are then diluted again into a microtiter plate and theprocess is repeated until the entire plate is derived from a singlecolony. This serial process typically requires 2-3 months to completeand usually allows only about 1000 different types of cells to bescreened for the desired functionality (e.g., producing a specificantibody).

Two factors determine the time required to isolate a single monoclonalhybridoma by this method. First, the sensitivity of the assay used todetect antibodies of interest sets the frequency at which cloned cellscan be tested for specificity—for example, sufficient concentrations ofantibodies that can be detected by enzyme-linked immunosorbant assays(ELISA) are achieved seven to ten days after seeding individual cellsinto a microtiter plate. Second, the total number of manipulationslimits the number of clones that can be screened efficiently in anysingle round of selection (10-100 plates/screen).

Two alternatives for sorting cells into microtiter plates at limitingdilutions include picking clones from semi-solid media, andfluorescence-activated cell sorting (FACS). Cells plated in agar orother hydrogels are challenged to survive and grow slowly, and thecorrelation between cells that stain positive in FACS and those thatreadily secrete products is not straightforward. Both methods haveimproved the efficiency of screening by serial dilution, but theresulting cultures usually require additional independent testing byELISA or equivalent methods to verify secretion and specificity. Othermethods have been developed for the analysis of individual cells inlarge numbers, such as microfluidic devices, cell-based microarrays,ELISPOT and hemolytic plaque assays, but these methods do not allow bothhigh-throughput analysis of a secreted product and the recovery ofliving cells for clonal expansion.

Generally, arrays of antibodies with known specificities are used todetect the presence or absence of specific analytes in an unknownmixture, which is used as an analytical tool for detecting knownantigens with known antibodies; it is not designed for discovering newtypes of antibodies. Arrays of microwells are also used for screeningfor hybridomas producing antigen-specific antibodies but then aresubsequently, not in parallel, screened by traditional immunoassays(flow cytometry, ELISA) for antigen specificity.

In contrast, examples of the methods, apparatus and kits describedherein may use a moldable array of microwells or chambers (e.g., ˜50-100microns in diameter) to retain one (or a few) cells in each microwell.The array is placed in physical contact with a substrate in such amanner that the microwells become closed containers or a test apparatus.Incubation of this system allows the cells to produce products, such as,antibodies, cytokines and other secreted products, that are thenimmobilized on the substrate in the regions contacted by the microwells.In this manner, a microarray of the cellular products from eachmicrowell is produced. After incubation of the system for a suitabletime, e.g., 1, 5, 30, 40, 50 minutes to a few hours, the microwell arrayis removed from the substrate, and the immobilized cellular products onthe substrate, the microarray or microengraving, may be screened with aknown species to determine whether or not the immobilized cellularproduct(s) associate with the known species. The method disclosed hereinrepresents a novel approach by combining detection of species present inan unknown mixture and, in a parallel and efficient manner, screeningfor hybridomas producing antigen-specific antibodies. Additional usesfor screening non-cellular products using the methods, apparatus andkits are also described.

The soft lithographic technique is used to microengrave a dense array ofmicrowells (0.1-1 nL each) containing individual cells to print acorresponding array of the molecules secreted by each cell. The cellsremain in culture in a microwell after the engraving, and themicroarrays are interrogated in a manner similar to commercialmicroarrays of proteins or antibodies—for example, by use offluorescently labeled reagents and laser-based fluorescence scanners.This method, therefore, enables rapid identification of those cells thatexhibit desired properties, such as secretion of an antigen-specificantibody, and their subsequent recovery from individual wells for clonalexpansion.

Referring to FIGS. 1A and 1B, a top view of a moldable slab 100 isshown. The moldable slab 100 comprises a plurality of microwells orchambers, such as microwells 110, 120 and 130. In the illustration shownin FIG. 1A, each of the microwells is shown as having substantially thesame cross-sectional shape. For example and also referring to FIG. 1B,which shows a cross-section through line 1B-1B in FIG. 1A, thecross-section of the moldable slab 100 shows microwells 110, 120 and 130as being circular when viewed from the top and generally cylindricalwhen viewed from a side. The exact number, dimensions, shape and thelike of each microwell of the moldable slab 100 may vary. For example,when viewed from the top, the cross-section of each microwell may becircular, square, elliptical, toroidal, rhomboid or other selectedshape. In addition, any particular microwell of the moldable slab may bea different shape than another microwell in the moldable slab.

In certain configurations, each microwell of the moldable slab may besized and arranged to retain or to hold a single cell or a few cells(e.g., 3-5 cells), such as a bacterial cell, a hybridoma or otherselected cell. In some examples, the diameter of each microwell of themoldable slab may vary from about 10 microns to about 100 microns, moreparticularly, about 25 microns to about 100 microns, e.g., about 50-100microns. Additionally each microwell is separated from another by alength similar to the depth and/or height. The size of any selectedmicrowell may vary depending on the size of the cell, or cells, to beretained by the microwell. In certain examples, the microwell is sizedto be large enough so that the cell may remain viable but is not solarge that any products produced by the cell will be diluted by a largefluid volume. For example, the volume of each microwell of the moldableslab is large enough to retain a cell and to provide a buffer,nutrients, etc. to keep the cell alive, but the volume of the microwellis not so large that any desired screening products will be diluted bysolvent or buffer to a non-detectable level. In certain configurations,the volume of the microwell varies from about 1 picoliter to about 100nanoliters, more particularly about 10 picoliters to about 10nanoliters, e.g., about 100 picoliters to about 1 nanoliter.

The exact number of the wells or chambers in the moldable slab may vary.In some examples, the moldable slab may include a single large microwellwhere a single species may be screened. For example, a moldable slab mayinclude a single type of cell, catalyst or other selected species thatmay be screened. In configurations where the moldable slab is configuredas an array, the number of individual microwells may vary from about 24,48, 96, 384, 1024, 2048, 5096 or more or any value in between theseillustrative values. As material in the moldable slab is transferred tothe substrate, an array of disposed material forms on the substratewhich reflects the material present in the microwell or microwells ofthe moldable slab. One or more of the microwells in the moldable slabmay be blocked or prevented from transferring material to the substrateusing an insert or device placed between a particular microwell in themoldable slab and the substrate. This feature provides for selectivedisposition of arrays of material on a substrate.

The moldable slab may be configured in a variety of manners. Forexample, the moldable slab is configured as a plate comprising one ormore microwells. The moldable slab may also be configured as a beadcomprising one or more microwells or a bead configured to retainmaterial on its surface. Any particular configuration for a moldableslab may be used provided that material on the moldable slab, orproducts produced by material on the moldable slab, may be transferred,at least to some extent, to a substrate.

The moldable slab is configured to receive an insert comprising one ormore openings. In certain examples, the insert comprises a plurality ofopenings. Referring now to FIGS. 1C and 1D, a moldable slab 150comprises an insert 160 which may be disposed on top of the moldableslab 150. The insert 160 may be permanently fixed or may be removable.In this illustration, the insert 160 comprises a plurality of openings,such as opening 162. As the moldable slab 150 is brought into contactwith a substrate, the openings in the insert 160 allow for transfer ofmaterial from the moldable slab 150 to certain areas on the substrate.As material is transferred from the moldable slab 150 to the substratethrough openings in the insert 160, an array is formed on the substratewhich reflects the number of openings, and spacing of the openings, ofthe insert 160. This configuration permits the moldable slab 150 to takethe form of a single large microwell which may be used to hold materialsuch as, for example, a hybridoma, a bacterial cell and the like. Themoldable slab contains one or more inserts and the insert is produced byusing the same or similar materials that are used to produce themoldable slab.

The material or materials used to produce the moldable slab includepolymeric and metallic compositions. In certain examples, the moldableslab may be made from two or more materials, only one of which mayimpart the moldable properties to the slab. In other examples, two ormore materials which are elastomeric may be used. Illustrative materialsinclude, but are not limited to, glass, plastic (including both rigidand soft materials), polystyrene, polycarbonate, poly(dimethylsiloxane)(PDMS), nitrocellulose, poly(vinylidene fluoride) (PVDF), metals such asgold, palladium, platinum, silver and alloys thereof, steel and mixturesof any of these materials. The rigidity of some materials, such aspolystyrene, would not allow for conformal contact, and thus sealing, ofthe microwells against a substrate for testing the specificity of theantibodies produced in a parallel. PDMS, however, is a suitable materialfor this technique because it is not toxic, it is gas permeable, and itis easily compressed to form a tight, but reversible, seal with a rigidsubstrate.

In other examples, sols or gels, e.g., agar, a hydrogel, matrigel, etc.may be used in the moldable slab. In some examples, the material to beassayed, or cells which secrete a material to be assayed, may beembedded, impregnated in or injected into the moldable slab. Forexample, a monolayer of cells is cultured on the moldable slab. In someexamples, cells are embedded in a hydrogel which is used to produce, oris coated on, the moldable slab. Additional materials suitable for usein the moldable slabs will be readily selected by the person of ordinaryskill in the art, given the benefit of this disclosure.

The moldable slab may include additives, fillers, insulators, growthfactors and the like. For example, the moldable slab may include one ormore materials which act as an insulator to assist in keeping asubstantially constant temperature for the materials, e.g., cells, inthe moldable slab. The moldable slab may also include antibiotics orother compounds which can reduce or prevent growth of unwanted organismssuch as, for example, bacteria or fungus. Such additional materials maybe included in the moldable slab, coated on the moldable slab, e.g.,only in the microwells or on the entire moldable slab, or may otherwisebe impregnated in the moldable slab to provide a desired result.

The moldable slab may be cast in a mold using suitable materialsdescribed above. Photolithographic and replica molding techniques may beused. The material is coated on a master mold, vapor deposed on a mastermold or added onto or in a master mold to provide a moldable slabcomprising a desired number of chambers. In some examples, a moldableslab configured as an array is produced using photolithography andreplica molding, from monolithic slabs of poly(dimethylsiloxane) (PDMS).For example, a layer of a photoresist is patterned on a suitablesubstrate, such as a 3 inch silicon wafer, to produce a master with apositive relief pattern of the moldable slab. A suitable material (e.g.,PDMS) is cast onto the master, cured and peeled away to provide themoldable slab. In other examples, a moldable slab is produced by castinga material and using a press, plate, punch, air or the like to providedepressions in the surface of the cast material prior to curing orhardening of the cast material. Generally, at least 50 replicas arecreated from a single mold with minimal wear. Additional methods forproducing moldable slabs suitable for use with the methods, apparatusand kits disclosed herein will be readily selected by the person ofordinary skill in the art, given the benefit of this disclosure.Illustrative methods for producing moldable slabs are described in moredetail in U.S. Pat. No. 6,180,239 and U.S. Pat. No. 6,776,094, theentire disclosure of each of which is hereby incorporated herein byreference in its entirety.

A substrate may be used with the moldable slab to provide a testapparatus. The configuration of the substrate may vary and typically thesubstrate is selected such that it can “mate” with the moldable slab toprovide a substantially fluid tight test apparatus. The ability of themoldable slab to flex, distort, bend, conform, etc. to a surface of thesubstrate assists in providing a substantially fluid tight testapparatus. In certain examples, the substrate may be a solid substrate,such as a glass or plastic slide, which may be placed on or in contactwith the moldable slab. Generally, the array was designed to fit withinthe boundaries of a 1″×3″ glass slide—a common format for microarrayreaders; variations in shape and spacing of individual wells were usedto encode their specific location within the array.

For example and referring to FIGS. 2A-2D, a moldable slab 200 mayinclude a material, such as a cell 210 in a fluid medium 220. Asubstrate 230 (FIG. 2B) may be disposed on the moldable slab 200 to formtest apparatus 250 (FIG. 2C). The test apparatus 250 is substantiallyfluid tight such that the test apparatus 250 may be oriented in anydirection without substantial loss of fluid. For example, the testapparatus may be flipped over (FIG. 2D) to permit products secreted orproduced by the cell 210 to settle, adsorb, become immobilized, etc. ona surface of the substrate 230 under gravitational force. While FIGS.2A-2D are shown with a cell 210 in the moldable slab 200, non-cellularspecies, such as proteins, catalysts, nanomaterials, etc. could insteadbe disposed in the moldable slab such that the proteins, catalysts,nanomaterials, etc. could be transferred to the substrate 230.Additional materials that may be used in the methods, apparatus and kitsdisclosed herein will be readily selected by the person of ordinaryskill in the art, given the benefit of this disclosure. Illustrativematerials that may be used to provide a substrate include, but are notlimited to, glass, plastic (including both rigid and soft materials),polystyrene, polycarbonate, poly(dimethylsiloxane), nitrocellulose,poly(vinylidene fluoride) (PVDF), metals such as gold, palladium,platinum, silver and alloys thereof, steel, mixtures of any of thesematerials, and other materials that may be used to provide a moldableslab.

The substrate may be coated with a composition or compound that acts toretain material transferred from the moldable slab. In some examples, anentire surface of the substrate may be coated such that material may beretained on the entire surface. In other examples, select areas of asurface may include a composition or compound that acts to retainmaterial transferred from the moldable slab. Selective coating mayassist in formation of a plurality of “spots” or “patterns” on thesubstrate that can be assayed. For example, a coating that acts toretain material may be disposed over a mask which has openings spaced asuitable distance from each other to provide an array.

As material is transferred from the moldable slab, the material may beretained by the array coating and can be subsequently screened againstone or more species. In certain examples, the composition or compoundmay act to adsorb the material, e.g., by trapping some portion of thematerial in a matrix by physisorption of material on the substrate mayoccur. Such physisorption may be, for example, adsorption of antibodiesdirectly onto the substrate, adsorption of proteins recognizing theconstant region of the antibody's structure (Fc portion), e.g., ProteinA or G, adsorption of secondary antibodies recognizing the constantregion of the antibody's structure (Fc portion), e.g., Goat anti-Mouse,or combination of proteins and antibodies, e.g., Protein G and secondaryantibodies. Overall, the region of microwells on the PDMS slab matchedregions of the microarray and the specificity of the antibody producedby the individual cells in wells could be determined from themicroarray. Materials suitable for retaining a desired material on asubstrate will be readily selected by the person of ordinary skill inthe art, given the benefit of this disclosure.

In other examples, the substrate may include a linking group which canreact with a portion of the material to assist in retaining material onthe substrate. In some examples, the surface of the substrate may bechemically modified. For example, the surface of the substrate mayinclude silanes on glass. Modification of the surface with silanescontaining free amine or carboxylic acid groups for linking secondaryantibodies to surface by NHS-ester activation and subsequent amidelinkages may be used, for example. Modification of surface with silanescontaining a free nitrile group for electrostatic capture of primary orsecondary antibodies (See Bioconj. Chem., 1999, vol 10 pp 346-353) mayalso be used. Thiols on a metal, such as gold, palladium, silver orplatinum, may be used. For example, modification of the surface withthiols containing free amine or carboxylic acid groups for linkingsecondary antibodies to surface by NHS-ester activation and subsequentamide linkages may be used. Modification of a surface with thiolscontaining a free nitrile group for electrostatic capture of primary orsecondary antibodies may be used. Covalent modification of exposedfunctional groups on a polymeric surface to cross-link secondaryantibodies to surface may be performed. Additional methods andcompositions for chemically modifying a surface of the substrate will beselected by the person of ordinary skill in the art, given the benefitof this disclosure.

The material in the moldable slab may be attached to the substrate usingmoieties or tags appended to secreted molecules. For example, covalentmodification of secondary antibodies with a chemical moiety recognizedby a second chemical moiety immobilized on the surface of the substratemay be used to immobilize the secondary antibodies to the substrate. Inan illustrative example, biotinylated antibodies along with streptavidinimmobilized on a surface of the substrate may be used. Peptide sequencesor proteins may be appended to the secreted molecule and used to retainthe secreted molecule on a surface of the substrate. Addition ofappended moieties to the secreted molecules is performed in a manner toavoid or minimize disruption of the native structure of the molecule toprovide a secreted molecule that has a structure as close as possible tothe native structure of the molecule. Moieties or tags are selected forappending to secreted molecules or other molecules disposed in amoldable slab.

Material is transferred from the moldable slab to the substrate suchthat a suitable amount is present to detect association. An effectiveamount of material will vary for different materials disposed on thesubstrate depending, for example, on binding constants, temperature,ionic strength, concentration, pattern size, etc. In some examples, aneffective amount provides at least a detectable signal after a labeledspecies associates with the material disposed on the substrate. Adetectable signal may vary depending on the technique or method used todetect association. For example, more material may be disposed on thesubstrate where colorimetric methods are used for detection, while lessmaterial may be disposed on the substrate where mass spectroscopy isused for detection. In certain examples, a sufficient amount of materialis disposed to provide a concentration of about 1 attomole/cm² to about1 micromole/cm², more particularly about 1 femtomole/cm² to about 100nanomoles/cm², e.g., about 10 femtomoles/cm² to about 10 nanomoles/cm².

In certain configurations which involve a cell or a few cells disposedin a microwell of the moldable slab, the total time required forperforming an assay is less than about 24-48 hours, more particularlyless than a day, e.g., less than about 12 hours. For example, becausethe volume of each microwell may be a few nanoliters or less, the timerequired for a cell to express and/or secrete a detectable amount ofmaterial may be only a few hours, e.g., less than about 8-12 hours.Expression and/or secretion of the material is typically the ratelimiting step in performing assays involving monoclonal antibodies andother materials. In many instances, association of the species, evenafter time is allowed to reach an equilibrium state, proceeds rapidlywhen compared to the time required to express the material. Thesignificant time savings provided by the methods, apparatus and kitsdisclosed herein provides for a substantial increase in throughput toscreen large numbers of unknown species against a known species. It is asignificant advantage that certain embodiments of the methods, apparatusand kits disclosed herein can reduce screening time from months to lessthan a day.

The moldable slab may be loaded with a selected material by placing thematerial in a fluid, such as water, a buffer, a solvent or the like, andadding a suitable amount of the dissolved or suspended material to themoldable slab. Some material disposed in the moldable slab will beretained in the wells or chambers of the moldable slab. Additionalmaterial may be suspended in fluid on top of the moldable slab, e.g.,not in the wells of the moldable slab. Such additional material mayoptionally be wicked away or removed using, for example, micropipets,filter paper, drying agents, air streams and the like, such that onlythe chambers or wells of the moldable slab retain material, such ascells, catalysts, etc. This process assists in distinguishing whichwells in the moldable slab secreted a molecule or compound disposed onthe substrate that associated with a known species, and provides forrapid recovery and further analysis of such molecule or compound fromthe moldable slab.

The methods, apparatus and kits described herein may be used to providea material on a substrate that can be tested for a desired bindingspecificity. The species which the material on the substrate may beexposed to can vary depending on the nature of the material disposed onthe substrate. For example, where the material disposed on the substrateis a monoclonal antibody, the monoclonal antibody may be exposed to anantigen, or series or antigens, to determine whether or not themonoclonal antibody may associate with the antigen. Where the materialdisposed on the substrate is a catalyst, a reactant may be exposed tothe catalyst to determine whether or not the catalyst may associate withthe reactant. Where the material disposed on the substrate is an enzyme,a potential enzyme substrate, or a potential enzyme substrate analog,may be exposed to the substrate to determine whether or not the enzymeand the potential enzyme substrate, a potential enzyme substrate analog,can associate with the enzyme disposed on the substrate.

Where the material disposed on the substrate is a cellular productproduced only in a bacterial cell after successful transformation, thecellular product may be disposed on the substrate and screened with aknown substance that binds to the cellular product to distinguishbetween bacterial cells that have a transformation product, e.g., aplasmid, phasmid, etc., and those that do not. In some examples, a cell,or a cell in a moldable slab, may be exposed to an antigen and anyresulting cytokine or cytokines, e.g., monokines, lymphokines, etc., canbe disposed on a substrate and screened against a species to determineif a particular type of cytokine is produced in response to antigenexposure. It will be within the ability of the person of ordinary skillin the art, given the benefit of this disclosure, to select suitablespecies to test for association with a material disposed on a substrate.

Referring to FIGS. 3A and 3B where a single microwell or chamber 305 ofa moldable slab 300 is shown, substrate 310 may be placed in fluidcommunication with the moldable slab 300 such that a substantially fluidtight seal is formed to retain cell 330 within test apparatus 305. Cell330 secretes cellular products 342 and 344 which become adsorbed to thesubstrate 310. Subsequent to adsorption of the cellular products 342 and344, one or more known species, such as species 352 and species 354, maybe exposed to the cellular products 342 and 344 for a sufficient periodto allow association between the species and the cellular products ifsuch association may occur. In this simple illustration, species 352associates with cellular product 342, while species 354 does notassociate with either of cellular products 342 or 344. Prior todetection, an optional washing step may occur to remove any excessspecies that do not associate with the products disposed on thesubstrate 310. Species 352 may contain a label, such as a fluorescentlabel, colorimetric label, etc. such that detection of the product342-species 352 complex may be performed. While the illustrative exampleshown in FIGS. 3A-3D is shown with reference to a cell, non-cellularspecies, such as catalysts, nanomaterials, etc. may also be identifiedusing similar methods.

One particularly useful application for the methods, apparatus and kitsdisclosed herein is to provide an array of unknown monoclonal antibodiesdisposed on a substrate. The generation of monoclonal antibodies havesignificant utility as research tools and are also highly valuable aspotential drug candidates. Efficient screening methods have limited thenumber of candidate antibodies produced for therapeutic purposes. Theuses of the methods, apparatus and kits disclosed herein can beextended, however, to use the array of secreting cells to assay theresponse of cells on a solid substrate and include screening anysecreted material of interest from any cell type.

Examples of secreted materials include, for example, cytokines,chemokines, and pathogens such as viruses. Examples of cell types thatcould be used include all classes of lymphocytes (e.g., B cells, Tcells) and other cells specializing in secretion (for example, liver orkidney cells). Known antigens may be added to the array to determinewhich monoclonal antibodies could bind to the antigen. The known antigenmay be added to each member of the array or different antigens may beadded to some members in the array. In a typical configuration, morethan one type of monoclonal antibody may be present at each array membersuch that the monoclonal antibodies may be screened batchwise todetermine if any of the monoclonal antibodies in any particular arraymember can associate with the antigen. Such batchwise screening providesfor rapid testing of a plurality of monoclonal antibodies. If one ormore monoclonal antibodies in any particular array member do bind to theantigen, the monoclonal antibodies can be singled out individually forfurther testing to determine which particular monoclonal antibody, ormonoclonal antibodies, in the array member can bind to the antigen.

Instead of using a moldable slab in the methods, apparatus and kitsdisclosed herein, alternative compositions may be used. For example, amembrane comprising a plurality of openings is placed on top of asubstrate such as a glass or plastic slide, a metal plate, a porousfilter material, or other rigid slab on which the membrane is incontact. In certain examples, the membrane may be disposed on thesubstrate and a substantially fluid tight seal may be formed between themembrane and the substrate. Referring to FIG. 4A, a membrane 400 isshown comprising a plurality of openings. In certain examples, themembrane may have a thickness 410 (FIG. 4B) of about 0.01 mm to about 1mm, more particularly about 0.02 mm to about 0.2 mm, e.g., 0.03 mm toabout 0.1 mm. In certain examples, the holes of the membrane may besized and arranged to retain one or a few cells. In other examples, theholes may be sized and arranged such that each hole may retain a fewnanoliters of fluid volume. This configuration would allow use in amanner similar to the wells of the moldable slab, but would have theadvantage that two surfaces could be patterned or spottedsimultaneously, or if the substrate surface was porous, the continuousexchange of nutrients through the back surface of the pattern transferelement may be permitted. Referring to FIG. 4C, a membrane 430 may beplaced in contact with a substrate 440. Cells, such as hybridomas,bacterial cells, etc., may be disposed in the openings of the membrane430, and products secreted by the cells may be adsorbed to the surfaceof substrate 440. Referring to FIG. 4D, a membrane 450 may be placedbetween a first substrate 460 and a second substrate 470. Cells withinopenings of membrane 450 may secrete products which can be disposed on asurface of substrate 460 and a surface of substrate 470. In embodimentswhere two or more substrates are simultaneously patterned using amembrane, it may be desirable to turn, invert or agitate thesubstrate-membrane-substrate assembly periodically to assist dispositionof material on both substrate surfaces.

One or more of the substrates may include a compound or molecule thatacts to retain secretion products on a surface of the substrate. One ofthe two substrates may also have a compound or molecule disposed on itssurface that is capable of activating or modulating the secretionproducts of a cell or cells contained in the wells. Additionalalternative configurations for disposing and/or transferring material toa substrate will be readily selected by the person of ordinary skill inthe art, given the benefit of this disclosure. In some examples, eachhole or opening of the membrane may be the same size and configurationas the other holes or openings in the membrane. In other examples, theholes or openings in the membrane may be sized differently such thatdifferential disposition of material is patterned onto the substrate. Incertain examples, the membrane may be produced using materials such as,for example, rubber, Teflon® polymer, polycarbonate,polydimethylsiloxane, and thermoset polymers. Additional materials, suchas the moldable slab materials discussed herein, may also be used.

Identification of positive associations is performed using numeroussuitable techniques including, for example, fluorescence, massspectrometry, or other analytical methods used in traditionalimmunoassays (e.g., colorimetric methods). For example, the speciesadded to the substrate may include a fluorescent label such that if thelabeled species added to the substrate associates with the materialdisposed on the substrate, fluorescence emission may occur. Illustrativefluorescent labels include, for example, fluorescein isothiocyanate,didansyl chloride, lanthanides and lanthanide chelates, Alexafluor®dyes, inorganic semiconductor nanocrystals (e.g., quantum dots composedof II/VI or III/V semiconductors), and similar labels. Any fluorescenceemissions may be detected visually or may be detected using suitableinstruments, such as fluorescence microscopes, fluorimeters, cameras, orinstruments that include a charge coupled device, a photomultipliertube, a diode array and the like. Other labels that emit light, e.g.,phosphorescent labels, chemiluminescent labels, etc., may also be usedand detected using similar techniques as those used in connection withfluorescence detection.

The detectable moiety added to the substrate may include a colorimetriclabel such that if the labeled species added to the substrate associateswith the material disposed on the substrate, and after addition of asuitable enzyme substrate, a color may result. For example, acolorimetric label is an enzyme, such as horseradish peroxidase. Afteran enzyme substrate, such as, for example, o-phenylenediaminedihydrochloride, is added to the enzyme a colored product is produced ifthe colorimetric label is present. The colored product may be detectedvisually or may be detected using suitable instruments such as, forexample, UV/Visible instruments, plate readers, etc. In some examples,the colorimetric label may be a dye, e.g., an organic or an inorganicdye, such that if association occurs, the well or chamber remainscolored, whereas if no association occurs, the well or chamber is clearand colorless. For example, if no association occurs the well appearsclear or nearly colorless after unassociated labeled species are removedby washing.

Other detectable markers include a radiolabel. The radiolabel may beintegrated into the species or may be added as a tag to the species.When a radiolabel is used, it may be desirable to construct thesubstrate with an absorbing material between array members to prevent orreduce crosstalk between the various members disposed on the substrate.Illustrative radiolabels include, but are not limited to, ³H, ¹⁴C, ³²P,³³P, ³⁵S and ¹²⁵I. The species disposed on the substrate may beradiolabeled, and upon association, any radioactive emission from thespecies may be quenched by a molecule or group which associates with thespecies disposed on the substrate. Suitable radiolabels for use in themethods, apparatus and kits disclosed herein will be readily selected bythe person of ordinary skill in the art, given the benefit of thisdisclosure.

Binding may be measured using mass spectroscopy. For example, thespecies may be allowed a sufficient time to associate and the contents(after optional washing steps) of a particular array member, or aselected array on a substrate, may be removed and analyzed using massspectroscopy. Numerous different mass spectroscopic techniques may beused. For example, matrix-assisted laser desorbed ionization (MALDI),electrospray ionization (ESI), fast atom bombardment (FAB), time offlight (TOF), MALDI/TOF, ESI/TOF, chemical ionization (CI), liquidsecondary ion mass spectroscopy (LSIMS) or other mass spectroscopictechniques may be used. In some examples, tandem mass spectroscopy maybe performed. Mass spectroscopic techniques are useful fordistinguishing between association and non-association. In exampleswhere mass spectroscopy is used, an array may be generated on anappropriate substrate (e.g., a metal plate for MALDI). The entire arraymay be probed with a mixture of proteins used for immunization (e.g.,entire cell lysates) and then characterized by mass spectrometry.Identification of proteins, lipids, or carbohydrates which are bound byspecific antibodies may be accomplished, for example, by comparing thespectrometry data against databases of known biomolecules or compounds.

A proximity assay may also be used. For example, the species disposed onthe substrate may be labeled with a radioactive label prior to transferto the substrate. The species added to the transferred species on thesubstrate may include a fluorescent label, such that if association ofthe two species occurs, radioactive emission will excite the fluorescentlabel, and fluorescence emission may be detected as a positive indicatorof association. Because this energy transfer process requires theradioactive label and the fluorescent label to be close, e.g., within afew microns, fluorescently labeled species that remain free in solutionwould not emit light. Such proximity methods have the added benefit thatno washing steps or separation steps are required to determine ifassociation occurs. Any fluorescence emission may be detected using theillustrative techniques disclosed herein, e.g., plate readers,flourimeters, etc. Suitable fluorescent and radioactive labels forperforming proximity assays to assess association will be readilyselected by the person of ordinary skill in the art, given the benefitof this disclosure.

A significant advantage of the methods, apparatus and kits disclosedherein that if association of the species does occur, then the materialin the moldable slab, e.g., the cell or cells in a particular well orchamber, may be recovered and used for further analysis. In contrast,many existing methods do not permit recovery of cells for further useand/or analysis but instead require re-isolation and/or regrowth of thecells. For example, because the methods, apparatus and kits disclosedherein permit the cells to remain alive in the wells of the moldableslab, it is possible to produce multiple arrays for screening bystamping or patterning the array onto multiple substrates, and toretrieve positively identified cells for clonal expansion bymicropipetting or similar techniques.

Cells confined in microwells and sealed against a glass slide (such thatthe total media available was limited to the volume of the microwell)may be maintained for about 5 hours and up to 24-48 hours, e.g., 1, 2,6, 12, 18, 24, or 36 hours without a significant loss to viability.(FIG. 10). When a moldable slab is removed from a substrate and immersedin media, the cells remained loosely adhered to the bottom of the wells;vigorous rinsing or intentional extraction of the cells was required todislodge them from the wells. Because the cells in the microwells remainviable after printing, the same set of microwells could produce multiplecopies of an engraved microarray at different time points, by Elispot,FACS, ELISA, or other immunochemical methods is challenging. Theresulting microarrays have a high degree of similarity, but are notidentical; variations between specific spots may result from eitherfluctuation in the rate of secretion related to various stages of celldivision or cell death.

The species (e.g., secreted cellular product), and/or cell producing thespecies, may be retrieved from the moldable slab using numeroustechniques. For example, a micropipette may be used. An array of wellsin the moldable slab can be coded or addressed to identify specificwells in the array to match positive hits from the substrate. Methodsfor coding the system include adjusting the spacing between wells, theshape of the wells, and the size of the wells. After removing the arrayof microwells from the substrate or the moldable slab, the wells can beimmersed in appropriate cell culture media to maintain the viability ofthe cells until testing and/or characterization is complete. Extractionof the desired cell(s) from the identified wells can be performed with amicropipette. The extracted cell(s) may be expanded to generate a stablecell line, e.g., a stable hybridoma cell line. Expansion may be carriedout in a microtiter plate (e.g., 96 or 384-well plate) containingsuitable media to sustain cell viability. For example, in the case of ahybridoma cell line, a population of adherent feeder cells (e.g.,fibroblasts) may be present to condition the media and provide a surfacefor cell-cell contact. Culturing the cells in the microwells for 1-3days prior to extraction by micropipette would allow a few cycles ofcell division within the microwells and make it possible to extract afew copies of the desired cell rather than a single copy. The exactconditions required to sustain the cells will vary depending on the celltype, and it will be within the ability of the person of ordinary skillin the art, given the benefit of this disclosure, to select suitablemedia and growth conditions to promote cell viability.

Microengraving offers three primary advantages over traditionalscreening by serial dilution and ELISA. First, microengraving allows theidentification and segregation of the cells that secreteantigen-specific antibodies from a polyclonal mixture early in thescreening process. The ability to isolate those cells should preserveslow-growing clones and rare clones (e.g., those that recognizeparticular epitopes of interest); traditional screening by serialdilution tends to favor fast-growing clones because the time required toattain detectable levels of antibodies is sufficiently long to allowoutgrowth of the population tested. Second, segregation of cells earlyin the screening process reduces the labor and time required to maintainmany individual clones while characterizing the antibodies produced forappropriate reactivity in immunochemical assays. Table 1, below,summarizes the significant differences in materials and time requiredfor cloning by microengraving and serial dilution. Table 2, below,summarizes the cost analysis for microengraving and limited serialdilution. Third, microengraving simplifies the requirements forscreening polyclonal populations to identify clones with differentspecificities. A single microarray can be screened with multiple,differentially labelled antigens, or fragments of antigens; equivalentscreens by ELISA would require independent assays for each conditiontested. The ability to immunize mice with mixtures of antigens furthersimprove the rate of selection. A current disadvantage of microengravingis the manual retrieval of cells from the microwells. Existing ormodified instruments for picking mammalian cells from colonies areuseful for automated retrieval of cells, thereby further reducing thetime involved in the screening process.

TABLE 1 Comparison of Time and Resources Required to Clone HybridomasLimiting Serial Dilution Microengraving Method Time after fusion untilfirst screen 7-13 days 7-10 days Number of wells (per plate) 96-384wells 25,000-100,000 wells Percent wells filled (average) 33-100% (1-2cells/well) 50-80% (1-3 cells/well) Number of cells (per plate) ~32-384cells ~12,500-80,000 cells Typical number of plates per screen  10-1001-10   Total volume of fluid per well 75-200 μL 0.1-1 nL Time tilldetectable levels of antibody 2-7 days 2-4 h are produced Antigenrequired per screen (per plate) 24-48 μg ~0.75 μg Use different antigensin a single No Yes, multiple labels screen? Time per screen (culture +assay) 7-10 days ~10 h Minimum screens to determine 2-3 rounds 1-2rounds monoclonality Minimum time for screening 19-30 days 1-2 days Timerequired to maintain cultures 5 min (every 2 days) 5 min between screens(per plate) Expected yield of desired hybridomas 0-1 0->>1 (per plate)

TABLE 2 Cost Analysis for Microengraving Limiting Serial DilutionMicroengraving Method Cost for Immunization of Mouse (1 antigen $120 (6wks) $120 (6 wks) requires ~1 mg total material) Time after fusion untilfirst screen 7-13 days 7-10 days Cost of screening/plate† Labor (sortingand screening) 1 hr FTE 1 hr FTE Actual Time (culture + assay) 5-7 days~10 h Supplies (plates and tips)  $8 $12* Reagents (antibodies &proteins) $6.50 + 24-48 mg Ag $0.50 + 1-7 mg Ag Media $12 $6 Total costper plate ($)   $26.50   $18.50 Number of wells (per plate) 96-384 wells25,000-100,000 wells Percent wells filled (average) 33-100% (1-2 50-80%(1-3 cells/well) cells/well) Number of cells (per plate) ~32-384 cells~12,500-80,000 cells Cost per cell screened $0.07-$0.83 $0.0002-$0.001Minimum screens to determine 2-3 rounds 1-2 rounds monoclonality Typicalnumber of plates per screen  10-100  1-10 Total cost for cloning ONEhybridoma   $530-$7,950 $18.50-$370  Minimum time for screening 19-30days 1-10 days (Cost should scale as ~1/n with increasing numbers ofrecovered clones per screen) Expected yield of desired hybridomas (per0-1   0->>1 plate) Maximum yield of unique monoclonal  1 ??? (>1)hybridomas (per plate) Test different antigens in a single screen? NoYes, multiple labels (2-16)

Microengraving enables similar assays for detecting a variety ofsecretions from large numbers of individual cells in a (semi)quantitative manner. The captured secretions represent the amountsproduced over a fixed period of time, and analysis does not requiremonitoring a short-lived event such as calcium flux. The method is notlimited to screening hybridomas derived from mouse splenocytes, but alsocan be applied to Epstein-Barr virus-transformed human B cells, and inprinciple, primary cells from peripheral blood or tissue. The methodsare useful in measuring other secreted products, such as cytokines, formonitoring an immunological response and for measuring the frequency ofcells within a population that produce a specific secreted factor indiagnostic assays. Multiplexed labeling, and the ability to generatemultiple engravings from the same array of microwells, thereby allowingtesting for the presence of multiple secreted products from singlecells.

A protein or antigen is immobilized on a substrate, and then a speciessecreted by a cell is added to determine whether or not such speciesassociates with the protein or antigen that is immobilized on thesubstrate. A labeled secondary antibody may then be added to probe forpositive capture of the species. The protein or antigen is disposed onthe substrate by transferal from a moldable slab or by disposal usingconventional techniques such as, for example, pipetting. The labeledsecondary antibody may be labeled with any of the illustrative labelsdisclosed herein, e.g., fluorescent labels, radioactive labels,colorimetric labels, etc. Detection may be accomplished using any of themethods disclosed herein, such as, for example, fluorescence, massspectroscopy, etc., depending on the selected label.

The methods, apparatus and kits disclosed herein may be used toimmobilize antibodies on monolayers of cells. For example, secretion ofantibodies from wells of a moldable slab onto monolayers of fixed andpermeabilized cells followed by treatment with a fluorescently labeledsecondary antibody can provide a method to characterize antibodies bystructural labeling within the cell. Optical microscopy would allowidentification of antibodies labeling specific organelles. For example,organelles such as cytoskeleton, endoplasmic reticulum, Golgi apparatusor other organelles may be labeled.

The species that can be identified using the methods, apparatus and kitsdisclosed herein may be further characterized using conventionaltechniques, such as immunoprecipitation, Western blots, or otherbiochemical analysis to identify the specific species. For example, themonoclonal antibodies may be sequenced. X-ray crystallography, NMRanalysis and the like may also be performed to characterize thestructure of the identified species. Additional suitable techniques forcharacterizing the species identified using the technology disclosedherein will be readily selected by the person of ordinary skill in theart, given the benefit of this disclosure.

Engineered organisms may be used with the methods, apparatus and kitsdisclosed herein. For example, a modified Ig locus may be introducedinto an animal (e.g., a D^(h)−/− or J^(h)−/− mouse) that includes agenetic encoded chemical structure for attaching antibodies secreted tothe solid slab. For example, a recombinant Ig molecule includes asortase signal peptide sequence onto the end of the Ig locus. Thisaddition would produce antibodies with the sortase signal peptideexpressed on the Fc portion of the antibody. Incubation of the cells ina media containing recombinant sortase enzymes on top of a substratecontaining immobilized peptidoglycans allows covalent attachment of theantibody to the peptidoglycans via transpeptidation. Anotherillustrative example would include addition of a fragment of aubiquitin-specific protease that includes the active catalytic site ontothe end of the Ig locus. This addition would produce antibodies with aknown reactive oligopeptide sequence expressed on the Fc portion of theantibody. Incubation of cells producing these labeled antibodies on topof a substrate containing an immobilized electrophile (e.g., vinylmethyl ester) would allow covalent attachment of the antibody to thesurface. Such modifications may also be performed in bacterial cells,viruses, insect cell lines and the like.

Certain specific examples are described below to illustrate further thenovel and inventive subject matter disclosed herein. The followingmaterials and methods were used to demonstrate the compositions andanalytical techniques described herein.

The examples using known hybridomas suggested that it should be possibleto identify rapidly cells producing antigen-specific antibodies within apolyclonal mixture and retrieve them for clonal expansion. Mice withpeptide-loaded H-2K^(b)/streptavidin tetramers, and generated hybridomasby fusion of splenocytes with NS-1 cells were immunized according tostandard protocols. Following bulk selection of the fused cells,polyclonal mixtures were loaded into arrays of microwells and incubatedon glass slides coated with goat-anti-mouse IgG antibodies. Theresulting microarrays were stained using tetramers of H-2K^(b) preparedwith fluorescently-labeled streptavidin (FIG. 16A and FIGS. 17A-B).Approximately 200,000 cells from the mixtures were screened across tenmicroarrays. 50 cells were arbitrarily selected to extract for expansionfrom the ˜4,300 positive spots identified on the microarrays. The cellsexpanded in the microwells for four days (doubling every ˜12-24 h)before being extracted using a micromanipulator and deposited into a96-well plate. 42 clones survived extraction and subsequent expansion;of these clones, the supernatants of 17 of which showed strong responsesby indirect ELISA relative to a control antibody (AF6-88.5).

The specificity of the antibodies from four clones (c113, c127, c128,and c136) was tested further by the immunoprecipitation of properlyassembled H-2K^(b) molecules from detergent extracts prepared from³⁵S-methonine/cysteine-labeled EL4 cells (FIG. 16B). Three of the fourclones recovered H-2K^(b). No detectable amounts of IgG were present inthe supernatants of the clone that did not recover H-2K^(b) (c113),suggesting that this clone may represent a false positive selected fromthe combination of the microarray screen and indirect ELISA, or that ithad lost its ability to produce the antibody of interest (chromosomeloss or epigenetic change). The antibodies produced by clones 127, 128,and 136 were all IgG₁γ. SDS-PAGE of denatured ³⁵S-labeled antibodiesproduced by clones 127 and 136 showed single bands that migrateddifferently for both the heavy and light chains; these data suggest thatthe hybridomas produce unique antibodies. To further evaluate clone 136,the cells were expanded, frozen, revived, and then used to prepare anengraved microarray (FIG. 16C and FIGS. 17A-B). Antibodies were capturedon a slide coated with goat-anti-mouse IgG, and the array was probedwith both fluorescent antigen (H-2K^(b)) and a fluorescent antibody(goat-anti-mouse IgG). This array showed that all cells in thepopulation tested produced IgG that was also specific to H-2K^(b).

Cell culture and purification methods are more specifically describedherein. Cell types EL4, NS-1 (ATCC), HYB 099-01 (Statens Serum Institut,Copenhagen, Denmark) and Y3 cells were grown in Dulbecco's ModifiedEagle Medium (DMEM) (Gibco, Grand Island, N.Y.) supplemented with 10%(v/v) fetal calf serum (FCS, Hyclone, Logan, Utah), 50 unitspenicillin/50 μg streptomycin, 20 mM HEPES, 50 μM 2-mercaptoethanol, 1mM sodium pyruvate, and 0.1 mM nonessential amino acids (Gibco, GrandIsland, N.Y.) at 37° C. (5% CO₂). The cells were split every 2 to 3 daysunder sterile conditions.

Peptides, having the sequences SIINFEKL (SEQ ID NO.:1) and SIYRYYGL (SEQID NO.:2), were synthesized by standard Fmoc-based solid-phase peptidechemistry, confirmed by MALDI-MS and LC/MS analysis, and were useddirectly, without further purification. Murine β₂-microglobulin as wellas a fusion protein of murine class I MHC heavy chain having aC-terminal biotinylation sequence were individually expressed, purifiedand reconstituted to H-2K^(b) complexes. The recombinant proteins wereexpressed in Escherichia coli employing theisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible pET vector system(Novagen) and BL21(DE3) as an expression host. The recombinant proteinswere isolated as inclusion bodies and dissolved under denaturingconditions (8 M of urea). Reconstitution was performed under diluteconditions in the presence of a large excess of the appropriate peptide.Subsequently, the monomeric, soluble H-2K^(b)-peptide complexes wereappended with a biotin moiety under the agency of BirA biotin ligase(Avidity) and purified by size exclusion chromatography (Superdex 75,Amersham Biosciences) to remove aggregates and free biotin. Forimmunization, tetrameric complexes were produced through stepwiseaddition of the appropriate soluble MHC class I complex to Streptavidin(Invitrogen) to a final molar ratio of 4:1. For interrogation of themicroarray, tetrameric complexes were formed in a similar manner usingStreptavidin Alexa 546 (Invitrogen) or Streptavidin Alexa 647(Invitrogen).

Balb/c female mice (8 weeks-old, Taconic) were immunized once every twoweeks subcutaneously with an emulsified 1:1 mixture of antigen (25-35 μgdissolved in 100 μL PBS) and complete Freund's adjuvant (firstimmunization only) or incomplete Freund's adjuvant (Sigma-Aldrich, St.Louis, Mo.). Three days prior to tissue harvest, antigen (100 μgH-2K^(b)/tetramers in PBS) was injected intraperitoneally. The fusion ofsplenocytes with NS-1 cells was performed according to standardprotocols. Media used for bulk selection were supplemented with 20%(v/v) FCS (Hyclone, Logan, Utah) and 10% (v/v) Hybridoma Cloning Factor(Bioveris, Gaithersburg, Md.), hypoxanthine aminopterin thymidine (HAT;ATCC, Manassas, Va.), hypoxanthine thymidine (HT; ATCC, Manassas, Va.),and 50% PEG (Sigma-Aldrich).

Example 1 Microengraving

To prepare the microwells for engraving, cells were deposited on thesurface of the PDMS slab at an appropriate dilution and allowed tosettle before removing excess media, as shown in FIGS. 13A-B. The numberof cells deposited per well depended on the concentration of cells, thevolume applied, the time allowed for the cells to settle, the size ofthe microwells, and the size of the PDMS slab. (FIG. 6A). For slabs ofPDMS ˜25×50×5 mm³ containing 25,000 wells (100 μm diameters separated by100 μm), 0.5 mL of a cell suspension, ranging from 1×10⁵ to 5×10⁵cells/mL, deposited for three to five minutes yielded one to three cellsin ˜50-75% of the wells (FIG. 5). The percentage of wells filled (FIG.7) and the average number of cells per well (FIG. 8) were determined bycounting the amount of cells in randomly determined fields with a 10×lens and averaging the data collected from each microwell array.Referring to FIG. 7, as the concentration of cells present increased,the percentage of wells in the microarray filled by cells alsoincreased. Referring to FIG. 8, as the concentration of cells presentincreased, the average number of cells per well also increased.Referring to FIG. 9, the results observed were consistent with usinghigher concentration of cells to increase the average number of cells ineach well and using lower concentrations of cells to favor one or a fewcells in each well. Cells confined in microwells and submerged in alarge reservoir of culture media divided normally every 12-24 h for morethan one week in culture.

Given the established rate of immunoglobulin (Ig) secretion for plasmacells and their derivatives (5,000 molecules/s), a single cell confinedin a small volume (˜0.1-1 nL) produced detectable concentrations ofantibodies (˜0.1-1 μM) in less than 5 h. Two established hybridomas—Hyb099-01, anti-chicken ovalbumin, and Y3, anti-mouse H-2K^(b) (majorhistocompatibility complex (MHC) class I) —were used to test thefeasibility of the method for engraving microarrays of secretedantibodies on a glass slide. The antibodies secreted were detected intwo ways (FIGS. 14A-E). In the first approach, secondary antibodies,goat anti-mouse Ig (gamma), were immobilized covalently onepoxide-functionalized slides; these slides were then incubated with anarray of cells for 2 h, and interrogated with a mixture of fluorescentlylabeled antigens. Correlating the region of microwells on the PDMS slabwith the matching region of the microarray data showed that (1) thefluorescent spots corresponded to the wells containing cells, (2) thelevel of non-specific binding of the fluorescently labeled proteins waslow in regions where there were empty wells, and (3) the specificity ofthe antibody produced by the individual cells in wells could bedetermined from the microarray. (FIGS. 14A-C and FIGS. 12A-C).

In a second approach similar to indirect ELISA, antigen—in this case,ovalbumin—was immobilized on slides by covalent attachment or bynon-specific adsorption, the primary antibody was captured from cellscontained in microwells, and the microarray was stained with afluorescently labeled secondary antibody (goat anti-mouse IgG) (FIGS.14D-E and FIGS. 11A-B). This format of the assay showed greatersensitivity to variations in the number of cells per well, and theamount of antibody secreted by individual cells, than the one usinglabeled antigens (FIG. 14A). The presence (or absence) of successfulcomplexes formed between antibody and specific antigens was determined,and the relative rates of production by individual cells was assessed.

Cells confined in microwells and sealed against a glass slide (such thatthe total media available was limited to the volume of the microwell)showed little or no loss in viability for up to 5 h. (FIG. 10). When aPDMS slab was removed from a glass slide and immersed in media, thecells remained loosely adhered to the bottom of the wells; vigorousrinsing or intentional extraction of the cells was required to dislodgethem from the wells.

Determining the time-dependent variability of secretion from singlecells by Elispot, FACS, ELISA, or other immunochemical methods ischallenging. Because the cells in the microwells remain viable afterprinting, the same set of microwells were used to produce multiplecopies of an engraved microarray at different time points. A set ofmicrowells were incubated on a glass slide and was coated with secondaryantibodies for 2 h. The microwells were removed and rinsed gently withfresh media, and the microwells were incubated on a second glass slidefor another 2 h. (FIGS. 15A-B). The minimum incubation period is about 1min to about 10 min, e.g., 30, 45 sec, 1, 2, 3, 5, 8 min. Qualitativeinspection of the resulting microarrays indicates that they have a highdegree of similarity, but are not identical; variations between specificspots may result from either fluctuation in the rate of secretionrelated to various stages of cell division or cell death. The totalnumber of replicas that can be made is about 100, more particularlyabout 4 to about 10 replicas using the same set of cells.

Microarray fabrication and methods for rapid high-throughput screeningare described herein. The microwell arrays were fabricated inpoly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning) usingphotolithography and replica molding. One layer of photoresist(SU-8-100, Microchem, Newton, Mass.) was patterned on a 3 inch siliconwafer using a transparency photomask (CAD/Art Services, Bandon, Oreg.)to produce a master with a positive relief pattern of the microwellarray. To facilitate removal of PDMS in subsequent steps, the masterswere silanized by treatment with(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (UCT, Bristol,Pa.) in a vacuum desiccator for 1 h. PDMS was cast onto the master,cured for 2 h at 60° C., and peeled away. The PDMS was allowed to swellin hexanes for 24 h, deswell in acetone for 24 h, and then dry in anoven at 130° C. for 24 h to remove low-molecular weight oligomers. Themicrowell array was treated with an oxygen plasma (PDC-32G, Hayrick,Ithaca, N.Y.) for 20 seconds; this process also sterilizes the array.The plasma-treated device was immersed in a solution of 10% w/v BovineSerum Albumin (BSA) (0.01% sodium azide) for 1 h at room temperature andthen rinsed with sterile phosphate buffered saline (PBS, Gibco, GrandIsland, N.Y.). The estimated variation in height is less than 5%, thewells in the center of the array are taller than those on the outer edgeof the array. The phase contrast images indicate the lateral dimensionsvary less than 2%, the top of a single well is wider than the bottom ofthe same cell.

Glass slides (1″×3″, VWR brand) were prepared and cleaned in “piranha”solution (conc. H₂SO₄: 30% H₂O₂, 7:3) at 70° C. for at least 1 hour. Theslides were thoroughly rinsed with deionized water (Millipore, 18 MΩ)and immersed in an ethanolic solution containing3-glycidoxypropyltrimethoxysilane (95% ethanol, 1% v/v silane, pHadjusted to 4.5 using glacial acetic acid) at room temperature. Theslides were removed after 1 hour, rinsed twice in 95% ethanol, and driedin an oven at 130° C. for 12 h. A solution of antigen (10-100 μg/mLovalbumin (Sigma-Aldrich) or K^(b)-streptavidin tetramers,) or secondaryantibody (200 μg/mL Goat anti-mouse Ig (gamma) Zymed, San Francisco,Calif.) in PBS was deposited on the surface of a slide under a coverslip(LifterSlip, Erie Scientific Company, Portsmouth, N.H.) and incubatedovernight at 4° C. Following incubation, the slides were immersed inblocking buffer (PBS, 0.01% w/v NaN₃, 1% w/v BSA) for 1 h at 25° C. orstored overnight at 4° C. The slides were rinsed in PBS/Tween 20 (0.05%w/v, PBST), PBS, and then deionized water. They were spun dry for 5 minat 750 rpm immediately before being sealed against the microwell arrays.

A suspension of cells was then diluted to 1×10⁵ cells/mL inserum-containing media and 0.5-1 mL was pipetted onto the surface of themicrowell array. The cells were allowed to settle for 3 minutes. Thesurface of the array was dewetted by applying a piece of extra thickfilter paper (Bio-Rad, Hercules, Calif.) or by vacuum aspiration at oneedge of the array while tilting the array. The percentage of wellsfilled and the average number of cells per well were determined bycounting the number of cells in randomly-determined viewing fields witha 10× lens and averaging data collected from multiple microwell arrays.

To engrave the microarray, an array of microwells filled with cells anddewetted of excess media was placed well-side-down on the surface of atreated, dry glass slide. The combination of the array and glass slidewas sandwiched together in a hybridization chamber (DT-1001, Die-Tech,San Jose, Calif.); the screws used to clamp the chamber together weretightened just until finger-tight. The entire assembly was incubated at37° C. for 2-4 h. After incubation, the treated glass slide was removedfrom the surface of the microwell array and immediately immersed inblocking buffer (1% BSA/0.05% Tween 20/PBS), and agitated for 1 h atroom temperature. After placing the glass slide in blocking buffer, themicrowell array was quickly immersed in a bath of pre-warmed mediabefore media contained in the microwells completely evaporated. (FIGS.6A-E)

After blocking in preparation for interrogation of the microarray, glassslides were rinsed with PBST, PBS, and then deionized water for 5 mineach; the slides were spun dry for 5 min at 750 rpm. A solution ofeither goat anti-mouse secondary antibody (Alexa Fluor 488 or 532,Invitrogen) or fluorescent antigen (e.g. 10 μg/mL Ovalbumin-Alexa Fluor488 or 555 conjugate (Invitrogen) or K^(b) tetramers prepared usingstreptavidin-Alexa 546 or 647 (Invitrogen) in PBS (80 μl) was depositedon the surface of a slide under a coverslip (LifterSlip, Erie ScientificCompany, Portsmouth, N.H.) and incubated in the dark for 1 h at roomtemperature. The slides were rinsed with PBST, PBS, and deionized water,and spun dry for 5 min at 750 rpm. The slides were imaged with a GenePix4000B microarray scanner (Molecular Devices, Sunnyvale, Calif.) using532 and 635 nm lasers and factory-installed emission filters. The laserswere used at 100% power and the PMT gain was set between 600 and 900 tomaximize the dynamic range of the detector without saturation. Images ofthe microarrays were analyzed using GenePix Pro 6.1 (Molecular Devices,Sunnyvale, Calif.). Color ratio images were generated in GenePix andsaved in the red and green channels of a 24-bit TIFF file. Backgroundintensities were subtracted using median values measured in regionsbetween individual spots of the array. The signal-to-noise ratio for agiven positive spot in the array to negative (or background) spotswithin the same subarray was calculated by dividing the sum of themedian intensity values from each channel for a given spot by theaverage sum of median intensity values for the negative spots determinedfrom at least 20 negative spots. The mean values reported are theaverage of at least 128 positive spots from more than 12 subarrays.

In order to perform microscopy and micromanipulation, phase contrastimages were acquired using Metamorph software (v6.3r3, MolecularDevices, Sunnyvale, Calif.) and an inverted microscope (Nikon EclipseTE2000-E) equipped with a Hamamatsu ORCA AG camera. Cells were retrievedfrom individual wells using a micromanipulator (IM-9A, Narishige, Tokyo,Japan) fitted with hand-drawn capillaries (GC-1). To withdraw thecontents of a well, the array of microwells was positioned on themicroscope under a layer of media (˜1 mL), and a capillary with an outerdiameter of 100 μm (inner diameter ˜50 μm) was positioned directly overthe top of an appropriate well. A small volume (˜1-5 μL) was withdrawnwith the affixed syringe until the cells were removed from the wellsuccessfully. The tip was then transferred into a well of a 96-wellplate containing 200 μL media (10% hybridoma cloning factor) and thecell(s) expelled into the volume. Both extraction from the microwell anddeposition of the cells into another container (96-well plate) weremonitored visually to ensure the transfer of the cells into and out ofthe tip.

Example 2 Identification of Antibodies Specific for Surface Epitopes ofInfectious Agents

The methods described herein are useful to identify antibodies reactiveagainst surface-exposed antigens present on infectious agents (bacteria,viruses, fungi), e.g., i) to identify new therapeutic agents for use inpassive immunizations and ii) to discover candidate antigens for thedevelopment of new vaccines intended to invoke a protective humoralimmune response. Memory B cells from convalescent human patients forother diseases, e.g., Aspergillus and malaria, are immortalized andtheir secreted products are screened using the engraved microarray. Aschematic illustration (FIG. 18) of the method for identifyingantibodies that bind surface-expressed epitopes on a pathogen. B cellsare derived from an inoculated animal or a blood sample from aconvalescent patient and screening with different serotypes or mutantsof the pathogens should bias the screen for specific types ofantibodies—for example, serotype-independent ones.

Microarrays of antibodies from a polyclonal mixture of cells arescreened using whole pathogens; competitive assays using multipleserotypes or genetic variants allow systematic analysis of a range ofpathogens (bacteria, fungi, viruses). Subsequent characterization of theantigens recognized by identified antibodies can provide a catalog ofcandidate antigens for developing a vaccine against the agent. Theantibodies themselves are useful for passive immunization strategies.For example, target antigens are identified using a mouse model and ahuman pathogen, e.g., opportunistic fungal pathogen (Cryptococcusneoformans) that affects immunocompromised humans. Another strategyutilizes screening transformed populations of memory 13 cells fromconvalescent individuals. Translational research is used to assess valueof identified antibodies or epitopes for passive immunization ordevelopment of vaccines.

Using a mouse model for human disease, mice are immunized with fixed (orheat-inactivated) pathogens or inoculated with sub-lethal dosages byappropriate routes (e.g., inhalation, sub-cutaneous injection).Hybridomas are prepared from splenocytes bypoly(ethylene-glycol)-mediated fusion with mouse myeloma cells (NS-1,Sp2/0). After bulk chemical selection of the surviving hybridomas, thegrowing polyclonal populations are loaded into microwells and used toengrave arrays of polyclonal antibodies (FIGS. 13A-D). A uniqueadvantage that engraved microarrays afford over other methods forscreening antibodies against antigens (enzyme-linked immunosorbantassays, flow cytometry) is the ability to design the probes for thearray in a manner that biases the search for specific reactivity (FIG.19A). On arrays generated using hybridomas prepared from mice challengedwith C. neoformans, differentially-labeled variants of C. neoformans isused to identify two types of antibodies: 1) serotype-independentantibodies, and 2) antibodies recognizing antigens other than theglucuronoxylomannan layer (GXM) that masks much of the exposed surfaceon C. neoformans. For the first type, there are three serotypes of C.neoformans known to cause human disease (A, B, and C). Each variant islabeled with a different fluorophore (e.g., N-hydroxyl-succinimideAlexafluor 488, 532, or 647) and the array is incubated with asuspension of these yeast, which lead to some antibodies capable ofbinding all three serotypes.

In a second approach, drug-treated wildtype variants are used toidentify antibodies recognizing epitopes on the surface other than GXM.Common anti-fungal agents, echinocandins, can disrupt the formation ofthe GXM layer, and allow access to the underlying cellular surface.(FIG. 19B). Such antibodies could supplement the use of drugs to treatan infection by improving the ability of the host's immune system toclear cells lacking mannan layers.

Antigen-specific clones are identified and retrieved bymicromanipulation. The clonality and diversity of the antibodiesproduced are characterized by isotype, by SDS-PAGE gel electrophoresisof immunoprecipitated ³⁵S-labeled antibodies, and by genetic sequencing.Also, in vitro assays are used to determine the extent to whichindividual monoclonal antibodies, or oligoclonal mixtures, conferprotection via enhanced opsonization and phagocytosis of C. neoformansby macrophages or dendritic cells. A transgenic mouse that cannotproduce circulating immunoglobulins (AID−/−, μS−/−) provides abackground for measuring the usefulness of passive immunization in vivowithout convolving a host-derived antibody response.

The disclosed method, apparatus and kits identifies epitopes recognizedby neutralizing antibodies. The selected antibodies are those recoveredantibodies that recognize a specific protein from the pathogen byimmunoprecipitation or immunoblotting detergent-solubilized lysates ofthe bacteria, virus, or fungus. Specific staining with the monoclonalantibody and subsequent analysis by mass spectrometry allowsidentification of the protein recognized. Additional analysis usingsynthetic peptide libraries based on the sequence of the protein likelyis used to refine the identity of a specific epitope, i.e., finemapping.

In the event that an antibody binds the intact pathogen, but does notimmunoprecipitate or stain a protein from a lysate, the identity of theantigen is determined as follows. First, for viruses and yeast, thewhole microbe and corresponding lysates are treated with glycosidases,and subsequently, treated with the antibody to determine if binding isconserved after deglycosylation. This approach has led to successfulrecognition of carbohydrate epitopes essential for binding anti-HIVantibodies specific for gp120 (2G12 epitope). Second, for bacteria,identification of non-proteinaceous epitopes would require disruption ofpathways involved in surface-expression of glycolipids or othercell-wall components. For many bacterial pathogens (e.g., Salmonellaenteritidis), a range of mutant strains exist that are used to deducethe component recognized by an antibody.

Though mouse and humanized antibodies have had utility as therapeuticagents, it is increasingly understood that fully human antibodies inducefewer side effects than chimeric or transgenic ones. The method,described herein to identify and retrieve transformed human memory Bcells producing human antibodies. Blood samples from convalescentindividuals recovered from infectious diseases, e.g., Aspergillus,malaria, and influenza and transform the populations of human memory Bcells in these samples by incubation with Epstein-Barr virus and acocktail of suitable stimulants (CpG DNA, cytokines). Secreted human IgGby microengraving from such cells can be detected using the disclosedmethod, apparatus and kits (FIG. 20). These methods identify antibodiesfor use in passive immunizations, and identify what epitopes on apathogen instigate a humoral immune response.

Example 3 Simultaneous Determination of Phenotype and FunctionalBehavior of Individual Primary Cells

Another application for the disclosed method, apparatus and kits is thedetermination of the phenotype of a cell and its functional behavior(e.g., secretion of extracellular factors) using soft lithography formeasuring secreted factors from large numbers of single cells (>100,000)and for correlating them with the phenotypic markers displayed on theproducing cell. Traditionally, these characteristics for cells extractedfrom tissue or blood are established by independent methods—for example,flow cytometry and immunosorbant assays—that provide information abouteither phenotype or function, but rarely both. Furthermore, existingmethods for measuring functional behaviors, such as secretion ofcytokines, often require additional manipulations and culturing togenerate sufficient material from a bulk population of cells fordetection. The graphical abstract (FIG. 21) suggests how a softlithographic technique could provide both characteristics for a largenumber of cells. Comparison of the frequency of specific cells presentafter onset of a disease or administration of a vaccine would provide aprofile of how an individual is responding relative to others.Extraction of specific cells by micromanipulation would allow geneticsequencing of unique markers (e.g., B cell or T cell receptors).Allowing a single assay to measure both the phenotype and secretoryfunction of individual cells

The method, apparatus and kits described may be extended for identifyingand retrieving monoclonal hybridomas secreting antigen-specificantibodies so that a highly multiplexed platform for profiling largenumbers (>100,000) of primary cells is generated on the basis of bothsecreted factors and surface markers (FIG. 22). Minimally, four uniquesecreted factors and phenotypic markers, are realized and detected withan aim to detect at least 10. The experiments described here establishthe method using T and B cells from various tissues of mice, which canbe extended to analyze human lymphocytes derived from blood samples.

The method described herein utilizes the microengraving method,apparatus and kits described, however, the immunosorbant capture ofcytokines on the glass slide likely will require a sandwich-style formatin which an antibody immobilized on the surface captures the factor ofinterest and a second antibody, reactive against a different epitope, isused for labeling. Though cytokines are typically secreted from cells ata rate that is 1000-fold less than that for antibodies produced byhybridomas or plasma cells, a single cell confined in a microwell of50×50×50 μm³ generates sufficient cytokines for a concentration of 25ng/mL within 4 h. The nature of the assay requires immobilization of Ndifferent capture antibodies uniformly on the surface of the slide,where N is the number of secreted factors one aims to detect. Thisconstraint may reduce the signal available from each secretion by afactor equal to the number of different cytokines probed. For twocapture antibodies, the available surface area and reactive antibodiesis sufficient for detection (FIG. 23). The composition of depositionbuffers and testing alternative immobilization strategies can be alteredto maximize the number of available sites for capture.

Multiplexed detection using fluorescent materials is limited by thenumber of emitted wavelengths that can be cleanly distinguished from oneanother. For traditional organic dyes and standard optical filters, thelimit is approximately four. Two ways to extend this range are 1) theuse of quantum dots as labels, and 2) spectral imaging (or deconvolutionof overlapping signals). Quantum dots provide the optimal material fordetecting a multitude of cytokines. Conjugated polyclonal,affinity-purified antibodies are directed against different cytokines tocommercially-available quantum dots of various colors (Invitrogen orEvident Technologies) using N-hydroxylsuccinimide esters. Optimizationof the labeling conditions and subsequent analysis of cross-reactivityis tested using spotted arrays of recombinant cytokines captured onglass slides slabing appropriate mixtures of capture antibodies. IFNγ(Th1 response), IL-2 (activated lymphocytes), IL-13 (Th2 response), andIL-10 (regulatory cells) are measured.

Both hybridomas and mononuclear cells from blood remain loosely adheredto the bottom surface of the poly(dimethylsiloxane) (PDMS) microwellsduring manipulations of the wells. Because the cells can remain in thewells, standard protocols are used for immunofluorescence to analyzeextra- or intracellular proteins present on the cells. Scanning arraysof microwells containing labeled cells will require a microscopeequipped with an automated stage and focus. Commercial instruments forlaser-scanning cytometry on glass slides is suitable for the analysis,however, custom-designed optics can be tailored to allow the greatestdegree of flexibility in analysis.

An exemplary analysis is carried out as follows. Cells, e.g.,splenocytes from a mouse (e.g., C57Bl/6) are obtained and the number ofcells with specific phenotypic markers and their secretion patternsdetermined by microengraving. For the evaluation of immune responses,the focus is initially on T cell markers (CD4 and CD8), and twochemokines (IFNγ and IL-13). FACS analysis provides confirmation of thefrequency of cells. Intracellular staining for IFNγ and IL-13 gives anindication of the relative number of cells producing each of thosecytokines. Splenocytes taken from transgenic OTII mice, which have CD4+T cells expressing a T cell receptor specific for a peptide fragmentderived from ovalbumin are used as an example. Immunization of thesemice with ovalbumin, or cell cultures stimulated in vitro with theantigen and αIFNγ antibodies, skew the immune response to develop a Th1or Th2 response.

The example experiment allows confirmation of a hypothesis suggested bySallusto et al. that the age of a mature dendritic cell determines thelikelihood of an interacting CD4+ T cell to polarize into Th1- orTh2-type. (Langenkamp, A., Messi, M., Lanzavecchia, A. & Sallusto, F.Kinetics of dendritic cell activation: impact on priming of TH1, TH2 andnonpolarized T cells. Nat. Immunol. 1, 311-316 (2000)). Dendritic cellsfrom a donor mouse are isolated and exposed to ovalbumin in the presenceof lipopolysaccharide for 8-48 h. After loading with antigen, the cellsare transferred into an OTII mouse by intravenous tail injection. After1-2 days, the frequency of Th1- and Th2-polarized T cells present in thelymph nodes and the spleen are measured as a function of the maturationtime of the injected DCs. The DCs matured more than 12 h should induce agreater percentage of Th2-polarized or non-polarized T cells than DCsmatured for less than 12 h.

The data demonstrated the development and verification of the techniquefor connecting phenotype with functional responses for large numbers ofindividual primary cells. The application of the technology is useful toprofile immunological responses as a function of disease. It may be usedin the context of transgenic mice with model diseases (diabetes, mousepathogens), and to evaluate immune responses to human diseases, e.g.,opportunistic fungal pathogens (e.g., Aspergillus), malaria, influenza,and diabetes using blood samples. Because the cells can be retrievedfrom the microwells by micromanipulation, the technique may be extendedto include genetic sequencing of unique features from single cellsidentified in a screen, e.g., B cell or T cell receptors.

The application of the disclosed methods, apparatus, and kits willprovide a tool for monitoring an immune response directly by determiningthe efficacy of a vaccine or correlating the frequencies of particulartypes of cells upon onset of a certain disease. The ability to uselimited numbers of cells from a blood sample is particularly suited foruse as a diagnostic tool to detect early genetic defects in pediatrics(e.g., immunodysregulation, polyendocrinopathy, and enteropathy,X-linked syndrome (IPEX) —a deficiency of CD4+CD25+ regulatory T cells).The relative ease of processing a sample by this method allowsinexpensive diagnostic applications in clinical microlabs and/orthird-world countries. The methods are useful to characterize cellularidentity and behavior in patient-derived samples from individualssuffering from or at risk of developing infectious diseases, cancer, orneurological disorders.

Example 4 Engineering Antibodies to Improve their Orientation onSurfaces

To direct the orientation of cell secreted products relative to thesurface on which they are printed, a nucleic acid sequence encoding ashort peptide recognition sequence (5-15 residue, e.g, 8-10 residuesequence) is incorporated into a gene encoding the secreted polypeptide,e.g., the heavy and/or light chain of an immunoglobulin chain. Anenzymatic or other chemical reaction installs a unique (orthogonal)chemical moiety on the short peptide sequence that would provide aspecific site for attaching the secreted product, e.g., antibody, to asolid support or other scaffold (e.g., another molecule, enzyme, orpolymer matrix). The enzyme inducing the conversion or chemicalmodification is encoded in the cell itself or is provided externally,e.g., extracellularly in the culture medium. A transgenic mouse carryingthis modification is used to produce hybridomas containing this“chemical handle” without additional cloning or reengineering.

One example of such a peptide recognition sequence for a selected enzymeand the enzyme (BirA ligase). Another example is sortase, atranspeptidase encoded by many bacteria such as Staphylococcus aureus.Sortases recognize a 5-amino acid peptide motif, LPXTG (SEQ ID NO.: 3).Two other classes of enzymes that recognize short peptide motifs aretransglutaminases and lipoic acid ligases. The modifications are made toeither terminus of either the heavy or light chains, e.g., theC-terminus of the heavy or light chains contains the chemical tag.

Immunoglobulins (Ig) are engineered to contain specific chemical sitesfor the purpose of improving orientation and accessibility of bindingdomains in surface-based and nanoparticle-based assays. Transgenic micethat contain the sequence encoding the sites for chemical modificationare useful in making hybridomas that produce antibodies containingappropriate sites for defined ligation to surface-based assays or othermolecular constructs (drugs, labels) without requiring additionalcloning (FIG. 24).

The methods, apparatus and kits disclose a method for attachingantibodies to surfaces using a chemical functional group installed atthe C-terminus of the heavy chain of an Ig-gamma (IgG) (FIG. 25).Attaching an antibody modified in this manner to a surface directs itsorientation in a predictable manner, and thus improves functionality.For example, molecular cloning is used to insert a 15-amino acid peptidesequence (GLNDIFEAQKIEWHE (SEQ ID NO.: 4)) at the C-terminus of asecreted product such as an antibody. An enzyme, biotin ligase (BirA),produced by Escherichia coli ligates either biotin or a related ketoneanalog at the lysine residue in the peptide. Modification of the Igheavy chain with the ketone analog will allow site-specific attachmentusing a hydrazide moiety present on the surface. This approach is usedfor immobilizing antibodies on self-assembled monolayers (SAMs)supported on gold or palladium.

A stably-transfected cell line, is generated that expresses an antibodywith an appended amino-acid sequence at the C terminus of the heavychain. The variable regions of the heavy and light chains (IgG1, κ)expressed by a mouse hybridoma, Hyb 9901 (anti-chicken ovalbumin), frommRNA are then cloned. These fragments are subcloned into mammalianexpression vectors containing the constant regions for the heavy andlight chains. At the 3′ end of the sequence for the heavy chain asequence encoding a 7 amino-acid epitope is inserted, which recognizedby tobacco etch virus (TEV) protease (ENLYFQ/S (SEQ ID NO.: 5)where/indicates cleavage site) followed by the epitope recognized by theBirA ligase (GLNDIFEAQKIEWHE (SEQ ID NO.: 6)). Expression of theantibody is induced by transfection of the two plasmids into 293T or CHOcells. Proper assembly of secreted antibodies is verified by ELISA usingimmobilized ovalbumin.

BirA ligase from E. coli cultures is expressed and purified and then ananalog of biotin containing a ketone is synthesized according toreported protocols. To ligate the analog to the modified antibodiescollected from the cell cultures, a buffered solution of the antibodiesis incubated with BirA, biotin analog, and ATP. Successful modificationof the antibodies is confirmed by Western blot analysis using anti-mouseIgG to identify the heavy chain and anti-biotin antibodies (orstreptavidin).

SAMs are prepared on thin films (20 nm) of gold or palladium using amixture of two thiols, (1-Mercapto-11-undecyl)tri(ethylene glycol) and ahydrazide-terminated derivative, HS(CH₂)₁₁(OCH₂CH₂)₆OCH₂CO₂NHNH₂. Thedensity of sites for attachment are controlled by varying the molarratio of the two thiols used to prepare the SAM. Exposure of the surfaceto a buffered solution of the modified antibodies will lead to ligation(FIG. 25). The relative density of functional antibodies immobilized onthe SAMs is measured by surface plasmon resonance using SAMs withantibodies immobilized by standard protein coupling methods (e.g.,EDC-NHS ester) as controls.

The example described here establishes a method for attaching antibodiesto surfaces using a specific chemical ligation at the C-terminus of theheavy chain. Using this example, the modification of gold nanoparticlesor quantum dots with the engineered antibodies may be conducted. Atransgenic mouse incorporating the peptide tail at the C terminus of theIgG1 constant region is generated. Thus, all IgG1 antibodies generatedby hybridomas from these mice incorporate the specific handle fororiented attachment without additional cloning or modifications. Suchantibodies would have an intrinsic site for subsequent modifications(orthogonal labeling, monovalent ligation of a drug or enzyme).

When introducing elements of the examples disclosed herein, the articles“a,” “an,” “the” and “said” are intended to mean that there are one ormore of the elements. The terms “comprising,” “including” and “having”are intended to be open ended and mean that there may be additionalelements other than the listed elements. It will be recognized by theperson of ordinary skill in the art, given the benefit of thisdisclosure, that various components of the examples can be interchangedor substituted with various components in other examples. Should themeaning of the terms of any of the patents or publications incorporatedherein by reference conflict with the meaning of the terms used in thisdisclosure, the meaning of the terms in this disclosure are intended tobe controlling.

Although certain aspects, examples and embodiments have been describedabove, it will be recognized by the person of ordinary skill in the art,given the benefit of this disclosure, that additions, substitutions,modifications, and alterations of the disclosed illustrative features,aspects, examples and embodiments are possible.

What is claimed:
 1. An apparatus comprising a moldable slab reversibly sealed to a substrate surface, said moldable slab comprising an array of microwells, wherein each microwell has a side or diameter of less than 100 microns and contains not more than a single or a few cell(s), wherein at least one of said microwells contains the single or few cell(s), wherein the cell(s) are antibody-producing cell(s) that secrete monoclonal antibodies in a volume of 10 nanoliters or less of fluid in each said microwell containing the single or few antibody-producing cell(s); wherein the surface of the substrate facing each said microwell containing said single or few antibody-producing cell(s) is bound to monoclonal antibody secreted from said antibody-producing cell(s) in their respective microwell.
 2. The apparatus of claim 1 wherein each microwell has a diameter of 10 to 100 microns.
 3. The apparatus of claim 2 wherein each microwell has a diameter of 50 to 100 microns.
 4. The apparatus of claim 1 wherein the antibody-producing cell(s) secrete the monoclonal antibodies in a volume of 10 picoliters to 10 nanoliters.
 5. The apparatus of claim 1 wherein the antibody-producing cell(s) secrete the monoclonal antibodies in a volume of 1 nanoliter or less.
 6. The apparatus of claim 1 wherein the antibody-producing cell(s) secrete the monoclonal antibodies in a volume of 100 picoliters to 1 nanoliter.
 7. The apparatus of claim 1 wherein the substrate is a glass substrate.
 8. The apparatus of claim 1 wherein the moldable slab is made of poly(dimethylsiloxane).
 9. The apparatus of claim 1 wherein the surface of the substrate is coated with a protein that recognizes the constant region of an antibody's structure, which protein is bound to said monoclonal antibody secreted from said antibody-producing cell(s).
 10. The apparatus of claim 9 wherein the protein is Protein A.
 11. The apparatus of claim 9 wherein the protein is Protein G.
 12. The apparatus of claim 9 wherein the protein is a secondary antibody.
 13. The apparatus of claim 1 wherein the moldable slab is reversibly sealed to the substrate so as to form a substantially fluid tight seal.
 14. The apparatus of claim 1 wherein each microwell has a diameter of 10 to 100 microns; and the antibody-producing cell(s) secrete the monoclonal antibodies in a volume of nanoliter or less.
 15. The apparatus of claim 14 the moldable slab is reversibly sealed to the substrate so as to form a substantially fluid tight seal.
 16. The apparatus of claim 15 wherein the antibody-producing cell(s) secrete the monoclonal antibodies in a volume of 100 picoliters to 1 nanoliter.
 17. The apparatus of claim 1 wherein each microwell has a diameter of 10 to 100 microns; the antibody-producing cell(s) secrete the monoclonal antibodies in a volume of 100 picoliters to 1 nanoliter; the substrate is a glass substrate; the moldable slab is made of poly(dimethylsiloxane); the surface of the substrate is coated with a protein that recognizes the constant region of an antibody's structure; and the moldable slab is reversibly sealed to the substrate so as to form a substantially fluid tight seal.
 18. The apparatus of claim 1 wherein the substrate comprises a plastic material.
 19. The apparatus of claim 1 wherein the substrate is substantially planar.
 20. The apparatus of claim 15 wherein the surface of the substrate facing said microwells is substantially planar. 